Nucleic acid sequencing method and system employing enhanced detection of nucleotide-specific ternary complex formation

ABSTRACT

Provided are methods and systems for detecting formation of nucleotide- specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/326,356, filed Apr. 22, 2016. The entire disclosure of this earlierapplication is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology.More specifically, the invention concerns nucleic acid sequencingtechnology.

BACKGROUND

Accurate sequence determination of a template nucleic acid strand findsmany important applications in genomic analysis, molecular diagnostics,and the like. Even identification of a single nucleotide base from amongalternatives at a known position can serve as the basis for analysis ofsingle nucleotide polymorphisms (i.e., “SNPs”). Detection of shortstretches of known nucleic acid sequence has been used for identifyingbacterial, fungal, and viral pathogens from clinical or environmentalsources. Detecting inherited or acquired genetic variants (e.g., changesor mutations) in known nucleic acid sequences also provides importantinformation, for example having to do with changed susceptibility tocertain medications. Finally, sequencing on the genomic scale depends oncorrect identification of millions of nucleotides that can be assembledinto one or more contiguous sequences. Each of these instancescritically depends on correct identification of individual nucleotides,one at a time, at different positions along a template nucleic acidstrand.

The human genome project was accomplished entirely by using conventionalfluorescent Sanger dideoxyribonucleotide sequencing techniques. Sincethat time, follow-on technologies have simplified the procedure forobtaining polynucleotide sequence information with the intention ofintegrating genomic analysis into routine clinical and industriallaboratory applications.

Alternative nucleic acid sequencing platforms include sequencing byhybridization, and sequencing-by-synthesis. In the first instance,detectably labeled strands of DNA are contacted under hybridizingconditions with an array of up to many thousands of defined probesequences immobilized to a solid support. Short duplexes formed in theprocedure can be detected and analyzed by a computer for assembly into acontiguous sequence. Sequencing-by-synthesis procedures have takenvarious forms and also have been successful. Automation and improvementsin the chemistry of labeled nucleotide addition, detection, andsubsequent chemical processing have revolutionized procedures foracquisition of raw sequence information. In some cases, only relativelyshort stretches of nucleic acid sequence are determined, but massivelyparallel processing provides the information needed to deduce nucleicacid sequence on the scale of a genome.

Generally speaking, there is a need for extended sequencing read-lengthwith improved base-calling accuracy across different sequencingplatforms. The present invention addresses this need.

BRIEF SUMMARY

In one aspect, the disclosure relates to a method of determining whethera test nucleotide is the next correct nucleotide including a basecomplementary to the next base in a template strand immediatelydownstream of a primer in a primed template nucleic acid. The methodincludes the steps of: (a) contacting the primed template nucleic acidwith a first reaction mixture that includes a DNA polymerase; (b)contacting the primed template nucleic acid from step (a) with a secondreaction mixture that includes the DNA polymerase and the testnucleotide; (c) measuring binding of the primed template nucleic acid tothe DNA polymerase of the first and second reaction mixtures at one ormore points during each of steps (a) and (b), respectively, but withoutchemical incorporation of the test nucleotide into the primer; and (d)determining whether the test nucleotide is the next correct nucleotideusing the measured binding results from step (c). According to onegenerally preferred embodiment, the test nucleotide can be a nativenucleotide that does not include any added fluorescent label, and step(c) does not include measuring differences in fluorescence or absorbancesignals from a conformationally sensitive dye that changes opticalproperties as the result of the DNA polymerase binding to the testnucleotide. According to another generally preferred embodiment, thefirst reaction mixture does not include the test nucleotide. Preferably,the method further includes the step of (e) chemically incorporating thetest nucleotide into the primer of the primed template nucleic acid byformation of a phosphodiester bond. More preferably, the method furtherinvolves repeating steps (a)-(e). For example, steps (a)-(e) can berepeated at least 50 times. Alternatively, the method can furtherinclude the step of (f) repeating steps (a)-(e) using a second testnucleotide in place of the test nucleotide, where the test nucleotideand the second test nucleotide are different from each other. In otherembodiments, where the first reaction mixture does not include the testnucleotide, the method further includes the step of (e) replacing thesecond reaction mixture with a third reaction mixture that includes areversible terminator nucleotide having a reversible terminator moiety,and a DNA polymerase different from the DNA polymerase of the secondreaction mixture. More preferably, the method further includes the stepof (0 incorporating the reversible terminator nucleotide into the primerby formation of a phosphodiester bond. Yet more preferably, the methodfurther includes the step of (g) removing the reversible terminatormoiety from the reversible terminator nucleotide that incorporated intothe primer. Still yet more preferably, the method further involvesrepeating steps (a)-(g) at least 50 times. In other embodiments, wherethe first reaction mixture does not include the test nucleotide, theprimed template nucleic acid can be immobilized to a solid support, andstep (b) can involve moving the solid support from a vessel containingthe first reaction mixture to a different vessel containing the secondreaction mixture. In other embodiments, where the first reaction mixturedoes not include the test nucleotide, the primed template nucleic acidcan be immobilized to a solid support, and each of steps (a) and (b) caninclude flowing reaction mixtures over the primed template nucleic acid.In other embodiments, where the first reaction mixture does not includethe test nucleotide, step (c) can include continuously measuring bindingof the primed template nucleic acid to the DNA polymerase during each ofsteps (a) and (b). In other embodiments, where the first reactionmixture does not include the test nucleotide, step (c) can includemeasuring optical signals indicating binding of the primed templatenucleic acid to the DNA polymerase and then calculating the differencebetween the measured optical signals to determine whether the measuredbinding resulting from step (b) exceeds the measured binding resultingfrom step (a). In other embodiments, where the first reaction mixturedoes not include the test nucleotide, step (c) includes measuringoptical signals indicating binding of the primed template nucleic acidto the DNA polymerase and then calculating the ratio of the measuredoptical signals to determine whether the measured binding resulting fromstep (b) exceeds the measured binding resulting from step (a). In otherembodiments, where the first reaction mixture does not include the testnucleotide, step (c) includes measuring optical signals indicatingbinding of the primed template nucleic acid to the DNA polymerase andthen calculating the time-dependent rate of change of the measuredoptical signals to determine whether the measured binding resulting fromstep (b) exceeds the measured binding resulting from step (a). In otherembodiments, where the first reaction mixture does not include the testnucleotide, the test nucleotide can be a native nucleotide selected fromthe group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In otherembodiments, where the first reaction mixture does not include the testnucleotide, the test nucleotide does not include an exogenousfluorescent label. In other embodiments, where the first reactionmixture does not include the test nucleotide, the test nucleotide can bean unlabeled test nucleotide that does not include an exogenous label.In other embodiments, where the first reaction mixture does not includethe test nucleotide, the test nucleotide is a nucleotide analog. Morepreferably, the nucleotide analog includes a reversible terminatormoiety. Still more preferably, the nucleotide analog that includes thereversible terminator moiety further includes a fluorescent label. Inother embodiments, where the first reaction mixture does not include thetest nucleotide, the primed template nucleic acid is immobilized to asurface, and step (c) involves measuring changes in refractive index tomeasure binding of the primed template nucleic acid to the DNApolymerase. More preferably, step (c) involves measuring changes inrefractive index by interferometry or surface plasmon resonance sensing.According to a different generally preferred embodiment, the DNApolymerase of the first reaction mixtures is free of added fluorescentlabel. According to a different generally preferred embodiment, the DNApolymerase of the first reaction mixtures is free of added fluorescentlabel that changes properties after the DNA polymerase binds to anucleotide. In other embodiments, where the first reaction mixture doesnot include the test nucleotide, and where the test nucleotide can be anunlabeled test nucleotide that does not include an exogenous label, theDNA polymerase of the first reaction mixture can be free of addedfluorescent label that changes properties after the DNA polymerase bindsto a nucleotide. More preferably, the primed template nucleic acid isimmobilized to a surface, and step (c) includes measuring changes inrefractive index to measure binding of the primed template nucleic acidto the DNA polymerase. According to a different generally preferredembodiment, the first reaction mixture further includes the testnucleotide at a concentration below that needed to achieve maximalternary complex formation when the test nucleotide is the next correctnucleotide, and the second reaction mixture includes the test nucleotideat a concentration sufficient to achieve maximal ternary complexformation when the test nucleotide is the next correct nucleotide.According to a different generally preferred embodiment, step (d)includes determining that the test nucleotide is the next correctnucleotide if the measured binding resulting from step (b) exceeds themeasured binding resulting from step (a).

In another aspect, the disclosure relates to a method of identifying thenext correct nucleotide including a base complementary to the next basein a template strand immediately downstream of a primer in a primedtemplate nucleic acid. The method includes the steps of: (a) contactingthe primed template nucleic acid with a first reaction mixture thatincludes a DNA polymerase; (b) contacting the primed template nucleicacid from step (a) with a second reaction mixture that includes the DNApolymerase and a first test nucleotide; (c) contacting the primedtemplate nucleic acid from step (b) with a third reaction mixture thatincludes the DNA polymerase, the first test nucleotide, and a secondtest nucleotide; (d) contacting the primed template nucleic acid fromstep (c) with a fourth reaction mixture that includes the DNApolymerase, the first test nucleotide, the second test nucleotide, and athird test nucleotide; (e) contacting the primed template nucleic acidfrom step (d) with a fifth reaction mixture that includes the DNApolymerase, the first test nucleotide, the second test nucleotide, thethird test nucleotide, and a fourth test nucleotide; (0 measuringbinding of the primed template nucleic acid to the DNA polymerase at oneor more points during each of steps (a)-(e), without chemicalincorporation of the first test nucleotide into the primer; and (g)identifying the next correct nucleotide as: (i) the first testnucleotide if the measured binding resulting from step (b) exceeds themeasured binding resulting from step (a), or (ii) the second testnucleotide if the measured binding resulting from step (c) exceeds themeasured binding resulting from step (b), or (iii) the third testnucleotide if the measured binding resulting from step (d) exceeds themeasured binding resulting from step (c), or (iv) the fourth testnucleotide if the measured binding resulting from step (e) exceeds themeasured binding resulting from step (d). According to one generallypreferred embodiment, the method further includes, after step (0, thestep of (h) chemically incorporating the next correct nucleotide intothe primer by phosphodiester bond formation. More preferably, each ofthe test nucleotides is different from the others, and each of the testnucleotides is selected from the group consisting of dATP, dGTP, dCTP,dTTP, and dUTP. Alternatively, each of the test nucleotides is differentfrom the others, and each test nucleotide is a native nucleotideselected from the group consisting of dATP, dGTP, dCTP, and dTTP.Alternatively, each of the test nucleotides is different from theothers, and none of the test nucleotides includes an exogenousfluorescent label. Alternatively, each of the test nucleotides isdifferent from the others, and where each test nucleotide is anucleotide analog including a reversible terminator moiety.Alternatively, the next correct nucleotide incorporated into the primerincludes a reversible terminator moiety. Preferably, the next correctnucleotide incorporated into the primer includes a reversible terminatormoiety. According to a different generally preferred embodiment, whenthe method further includes, after step (f), the step of (h) chemicallyincorporating the next correct nucleotide into the primer byphosphodiester bond formation, none of the test nucleotides includes areversible terminator moiety, and between step (0 and step (h) there isthe step of (i) removing any test nucleotide that bound to the primedtemplate nucleic acid, and then adding at least one nucleotide thatincludes a reversible terminator moiety. According to a differentgenerally preferred embodiment, when the method further includes, afterstep (f), the step of (h) chemically incorporating the next correctnucleotide into the primer by phosphodiester bond formation, thepolymerase used in steps (a)-(e) can be different from the polymeraseused in step (h).

In another aspect, the disclosure relates to a method of determiningwhether a test nucleotide is the next correct nucleotide including abase complementary to the next base in a template strand immediatelydownstream of a primer in a primed template nucleic acid. The methodincludes the steps of: (a) contacting the primed template nucleic acidwith a first reaction mixture that includes a first DNA polymerase; (b)contacting the primed template nucleic acid from step (a) with a secondreaction mixture that includes a second DNA polymerase and the testnucleotide, where the second DNA polymerase includes a label that isabsent from the first DNA polymerase; (c) measuring binding of theprimed template nucleic acid to the second DNA polymerase by detectingthe label present in a complex including the primed template nucleicacid at one or more points during steps (a) and (b), respectively, butwithout chemical incorporation of the test nucleotide into the primer;and (d) determining whether the test nucleotide is the next correctnucleotide using the measured binding results from step (c). Accordingto one generally preferred embodiment, the amino acid sequences of thefirst and second DNA polymerases can be the same. According to adifferent generally preferred embodiment, each of steps (a) and (b)occurs under buffer conditions that stabilize formation of ternarycomplexes, and that destabilize formation of binary complexes.Preferably, each of steps (a) and (b) occurs under high salt bufferconditions. Still more preferably, the first DNA polymerase does notinclude an exogenous detectable label. According to a differentgenerally preferred embodiment, the method further includes repeatingeach of steps (a)-(c) three times, with a different test nucleotide eachtime. Preferably, the label of the second DNA polymerase is afluorescent label. More preferably, step (c) does not include measuringa fluorescent signal from a conformationally sensitive dye that changesoptical properties as a result of the second DNA polymerase binding to acognate nucleotide. According to a different generally preferredembodiment, the method further includes removing the test nucleotide andany polymerase bound to the primed template nucleic acid. Preferably,there is the further step of contacting the primed template nucleic acidwith a third reaction mixture that includes a third DNA polymerase and areversible terminator nucleotide, and then incorporating the reversibleterminator nucleotide into the primer using the third DNA polymerase toproduce a reversibly terminated primer. More preferably, the methodfurther includes repeating steps (a)-(d) using the primed templatenucleic acid that includes the reversibly terminated primer in each ofsteps (a)-(c).

In yet another aspect, the disclosure relates to a method ofincorporating a nucleotide with a base complementary to the next base ina template strand immediately downstream of a primer in a primedtemplate nucleic acid molecule. The method includes the steps of: (a)contacting the primed template nucleic acid molecule with a firstreaction mixture that includes a DNA polymerase; (b) contacting theprimed template nucleic acid molecule from step (a) with a secondreaction mixture that includes the DNA polymerase and a first testnucleotide, without incorporating the first test nucleotide into theprimer; (c) measuring binding of the primed template nucleic acidmolecule to the DNA polymerase at one or more points during each ofsteps (a) and (b), without chemical incorporation of the first testnucleotide into the primer, to establish whether a ternary complexformed in step (b); and (d) selecting one of the following two options,(i) if it is established in step (c) that the ternary complex did notform in step (b), then contacting the primed template nucleic acidmolecule from step (b) with a third reaction mixture that includes theDNA polymerase in combination with the first test nucleotide and asecond test nucleotide, the second test nucleotide being different fromthe first test nucleotide; and (ii) if it is established in step (c)that the ternary complex formed in step (b), then contacting the primedtemplate nucleic acid molecule from step (b) with a fourth reactionmixture and performing an incorporation reaction to incorporate thenucleotide into the primer without first contacting the primed templatenucleic acid molecule from step (b) with the third reaction mixture.According to one generally preferred embodiment, the primed templatenucleic acid molecule is immobilized to a solid support. Preferably, thefourth reaction mixture includes a polymerase different from thepolymerase of the first and second reaction mixtures. Alternatively, thefourth reaction mixture includes a polymerase different from thepolymerase of the first and second reaction mixtures, and the nucleotideincorporated into the primer in step (d)(ii) includes a reversibleterminator moiety. According to a different generally preferredembodiment, the primed template nucleic acid molecule is immobilized toa solid support, and contacting steps (a) and (b) each include flowingthe reaction mixtures over the primed template nucleic acid moleculeimmobilized to the solid support. According to a different generallypreferred embodiment, the primed template nucleic acid molecule isimmobilized to a solid support, and contacting steps (a) and (b) eachinclude moving the solid support from a vessel containing the firstreaction mixture to a vessel containing the second reaction mixture.According to a different generally preferred embodiment, the first andsecond reaction mixtures each include the same concentration of thepolymerase. According to a different generally preferred embodiment, thefourth reaction mixture includes a polymerase different from thepolymerase of the first and second reaction mixtures. According to adifferent generally preferred embodiment, step (c) includes measuring byeither of interferometry or surface plasmon resonance sensing. Accordingto a different generally preferred embodiment, the first test nucleotideis a native nucleotide. According to a different generally preferredembodiment, the first test nucleotide includes a reversible terminatormoiety. According to a different generally preferred embodiment, thenucleotide incorporated in step (d)(ii) is any of a native nucleotide,and a nucleotide including a reversible terminator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a series of bar graphs indicating measured bindingsignals (vertical axis) as a function of interrogated dNTP identity(horizontal axis). The figure graphically illustrates a declining trendin measured signal for ternary complex formation (i.e., indicating thepresence of cognate nucleotide), while measured signal for binarycomplex formation (i.e., indicating the presence of non-cognatenucleotide) remains steady. Open bars indicate signals associated withcorrect positive calls, while stippled bars indicate signals associatedwith correct negative calls. Heavy diagonal fill indicates afalse-positive base call (T), and light diagonal fill indicates afalse-negative base call (C was missed).

FIGS. 2A and 2B are graphic traces illustrating binding activity(vertical axes) as a function of time (horizontal axes). Divisions shownin FIG. 2A indicate four parts to each complete cycle: binding,incorporation, quenching, and regeneration. FIG. 2B shows five parts toeach complete cycle, with the additional first (i.e., initial) partbeing due to the biphasic binding procedure that involved contactingwith polymerase and first (low) concentrations of nucleotide. Openarrows shown at cycle numbers 2, 4, 8, 10, 12, 16, and 20 indicatecorrect base calls; while solid filled arrows shown at cycles 6, 14, and18 indicate incorrect base calls. In both of the graphic traces, “cyclenumbers” identify independent steps for contacting primed templatenucleic acid with the indicated nucleotides.

FIGS. 3A and 3B present bar graphs that graphically illustrate measuredbinding signals (vertical axes) as a function of interrogated dNTPidentity (horizontal axes) in sequencing-by-binding procedures. FIG. 3Ashows results obtained using a standard sequencing-by-binding procedurethat employed only a single-phase examination step. Higher signalsindicated ternary complex formation, while lower signals were associatedwith binary complex formation. FIG. 3B shows results obtained using thebiphasic protocol for binding primed template nucleic acid withpolymerase and nucleotide in two phases. Open bars indicate signalsassociated with correct positive calls, while stippled bars indicatesignals associated with correct negative calls. One false-positive call(heavy diagonal fill) and one false-negative call (light diagonal fill)are indicated by arrows in FIG. 3A.

FIG. 4 is a graphic trace illustrating measured binding signal (verticalaxis) as a function of the progress parameter (horizontal axis).Durations (measured in seconds) of 12 progress intervals are shown abovethe plot. The first and second progress intervals representprimer/template loading and wash steps, respectively. The third progressinterval corresponds to the first phase (i.e., primed template nucleicacid interacts with polymerase in the absence of added nucleotide) ofthe biphasic protocol. Intervals 4-7, each lasting for 20 seconds,correspond to the second phases of the biphasic protocol (i.e., primedtemplate nucleic acid interacts with polymerase in the presence of addednucleotide). Nucleotide(s) present during progress intervals 4-7 were,respectively: dTTP; dTTP and dATP; dTTP, dATP, and dCTP; and dTTP, dATP,dCTP, and dGTP. The third nucleotide to be used in the combinationpromoted ternary complex formation, and corresponded to the cognatenucleotide (dCTP). Progress interval 8 was an EDTA wash step. Progressintervals 9-10 were, respectively, a regenerating wash preliminary toincorporation, and the incorporation step. Progress intervals 11-12 werean EDTA wash, and a regenerating wash preliminary to subsequentexamination.

DETAILED DESCRIPTION

Disclosed is a technique for detecting ternary complexes that include aprimed template nucleic acid molecule, a polymerase, and the nextcorrect nucleotide immediately downstream of the primer andcomplementary to the template strand of a primed template nucleic acid.Clear and unambiguous detection has been achieved despite interactionsbetween the polymerase and the primed template nucleic acid that promoteformation of nucleotide-independent binary complexes. The approachfollows from the observation disclosed herein that nonspecific binarycomplexes can be made to saturate on the primed template nucleic acideven in the absence of nucleotides. In contrast, nucleotide-specificternary complex formation exhibits dose-dependent binding and recruitspolymerase molecules in addition to those participating in binarycomplex formation. While not wishing to be bound by any particulartheory of operation, formation of nucleotide-specific ternary complexesand the nonspecific binary complexes may involve different interactionsbetween the polymerase and the primed template nucleic acid molecule.Only specific interactions leading to ternary complex formation areinformative with respect to the next correct nucleotide in thesequencing-by-binding technique.

The technique for enhancing detection of ternary complex formationinvolves comparing polymerase interaction with a primed template nucleicacid under binding conditions that differ by the presence or amount ofthe candidate nucleotide undergoing testing. For example, the testnucleotide (e.g., native dATP, dGTP, dCTP or dTTP; or nucleotideanalogs) can be present at two different concentrations while thepolymerase concentration is maintained substantially constant. The twotest nucleotide concentrations can be relatively lower and highercompared to each other (i.e., with the test nucleotide being includedunder both conditions). Alternatively, the two different bindingconditions may involve the presence and absence (i.e., zeroconcentration) of the test nucleotide while the polymerase concentrationis maintained substantially constant in all cases.

Advantageously, the technique can be practiced using various types ofnucleotides, including native (e.g., unlabeled) nucleotides, nucleotideswith detectable labels (e.g., fluorescent or other optically detectablelabels), or labeled or unlabeled nucleotide analogs (e.g., modifiednucleotides containing reversible terminator moieties). Further, thetechnique provides controlled reaction conditions, unambiguousdetermination of sequence, long read lengths, low overall cost ofreagents, and low instrument cost.

The disclosed technique can be applied to binding reactions used fordetermining the identity of the next base of a primed template nucleicacid by any means and for any reason. The technique can be used tomonitor specific binding of a DNA polymerase and the next correctnucleotide (e.g., a dNTP) complementary to a primed template nucleicacid, and to distinguish specific binding from nonspecific binding. Thetechnique may be applied to single nucleotide determination (e.g., SNPdetermination), or alternatively to more extensive nucleic acidsequencing procedures employing iterative cycles that identify onenucleotide at a time. For example, the methods provided herein can beused in connection with sequencing-by-binding procedures, as describedin the commonly owned U.S. patent application identified by U.S. Ser.No. 14/805,381 (published as US Pat. App. Pub. No. US 2017/0022553 A1),the disclosure of which is incorporated by reference herein in itsentirety.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. For clarity, the following specific terms have the specifiedmeanings. Other terms are defined in other sections herein.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedin the description and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the compositions, apparatus, or methods of thepresent disclosure. At the very least, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used herein, “sequencing-by-binding” refers to a sequencing techniquewherein specific binding of a polymerase and cognate nucleotide to aprimed template nucleic acid molecule (e.g., blocked primed templatenucleic acid molecule) is used for identifying the next correctnucleotide to be incorporated into the primer strand of the primedtemplate nucleic acid molecule. The specific binding interaction neednot result in chemical incorporation of the nucleotide into the primer.In some embodiments, the specific binding interaction can precedechemical incorporation of the nucleotide into the primer strand or canprecede chemical incorporation of an analogous, next correct nucleotideinto the primer. Thus, detection of the next correct nucleotide can takeplace without incorporation of the next correct nucleotide.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”or grammatical equivalents used herein means at least two nucleotidescovalently linked together. Thus, a “nucleic acid” is a polynucleotide,such as DNA, RNA, or any combination thereof, that can be acted upon bya polymerizing enzyme during nucleic acid synthesis. The term “nucleicacid” includes single-, double-, or multiple-stranded DNA, RNA andanalogs (derivatives) thereof. Double-stranded nucleic acidsadvantageously can minimize secondary structures that may hinder nucleicacid synthesis. A double stranded nucleic acid may possess a nick or asingle-stranded gap.

As used herein, a “template nucleic acid” is a nucleic acid to bedetected, sequenced, evaluated or otherwise analyzed using a method orapparatus disclosed herein.

As used herein, a “primed template nucleic acid” (or alternatively,“primed template nucleic acid molecule”) is a template nucleic acidprimed with (i.e., hybridized to) a primer, wherein the primer is anoligonucleotide having a 3′-end with a sequence complementary to aportion of the template nucleic acid. The primer can optionally have afree 5′-end (e.g., the primer being noncovalently associated with thetemplate) or the primer can be continuous with the template (e.g., via ahairpin structure). The primed template nucleic acid includes thecomplementary primer and the template nucleic acid to which it is bound.A primed template nucleic acid molecule can be extendable in apolymerization reaction or, alternatively, can have a blocking moietythat prevents extension.

As used herein, a “nucleotide” is a molecule that includes a nitrogenousbase, a five-carbon sugar (ribose or deoxyribose), and at least onephosphate group. The term embraces ribonucleotides,deoxyribonucleotides, nucleotides modified to include exogenous labelsor reversible terminators, and nucleotide analogs.

As used herein, a “native” nucleotide refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog. Examples of native nucleotides useful for carryingout the sequencing-by-binding procedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate)

As used herein, a “nucleotide analog” has one or more modifications,such as chemical moieties, which replace, remove and/or modify any ofthe components (e.g., nitrogenous base, five-carbon sugar, or phosphategroup(s)) of a native nucleotide. Nucleotide analogs may be eitherincorporable or non-incorporable by a polymerase in a nucleic acidpolymerization reaction. Optionally, the 3′-OH group of a nucleotideanalog is modified with a moiety. The moiety may be a 3′ reversible orirreversible terminator of polymerase extension. The base of anucleotide may be any of adenine, cytosine, guanine, thymine, or uracil,or analogs thereof. Optionally, a nucleotide has an inosine, xanthine,hypoxanthine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) or nitroindole (including 5-nitroindole) base.Nucleotides may include, but are not limited to, ATP, UTP, CTP, GTP,ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP,dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′, 3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP,ddATP, ddTTP, ddUTP and ddCTP).

As used herein, the “next correct nucleotide” (sometimes referred to asthe “cognate” nucleotide) is the nucleotide having a base complementaryto the base of the next template nucleotide. The next correct nucleotidewill hybridize at the 3′-end of a primer to complement the next templatenucleotide. The next correct nucleotide can be, but need not necessarilybe, capable of being incorporated at the 3′ end of the primer. Forexample, the next correct nucleotide can be a member of a ternarycomplex that will complete an incorporation reaction or, alternatively,the next correct nucleotide can be a member of a stabilized ternarycomplex that does not catalyze an incorporation reaction. A nucleotidehaving a base that is not complementary to the next template base isreferred to as an “incorrect” (or “non-cognate”) nucleotide.

As used herein, a “blocking moiety,” when used with reference to anucleotide analog, is a part of the nucleotide that inhibits or preventsthe nucleotide from forming a covalent linkage to a second nucleotide(e.g., via the 3′-OH of a primer nucleotide) during the incorporationstep of a nucleic acid polymerization reaction. The blocking moiety of a“reversible terminator” nucleotide can be removed from the nucleotideanalog to allow for nucleotide incorporation. Such a blocking moiety isreferred to herein as a “reversible terminator moiety.” Exemplaryreversible terminator moieties are set forth in U.S. Pat Nos. 7,427,673;7,414,116; and 7,057,026 and PCT publications WO 91/06678 and WO07/123744, each of which is incorporated by reference.

As used herein, a “test nucleotide” is a nucleotide being investigatedfor its ability to participate in formation of a ternary complex thatfurther includes a primed template nucleic acid (or blocked primedtemplate nucleic acid) and a polymerase.

As used herein, “polymerase” is a generic term for a protein or othermolecule that forms a ternary complex with a cognate nucleotide andprimed template nucleic acid (or blocked primed template nucleic acid)including but not limited to, DNA polymerase, RNA polymerase, reversetranscriptase, primase and transferase. Typically, the polymeraseincludes one or more active sites at which nucleotide binding may occur.Optionally a polymerase includes one or more active sites at whichcatalysis of nucleotide polymerization may occur. Optionally apolymerase lacks catalytic nucleotide polymerization function, forexample, due to a modification such as a mutation or chemicalmodification. Alternatively, the polymerase may catalyze thepolymerization of nucleotides to the 3′-end of a primer bound to itscomplementary nucleic acid strand. For example, a polymerase catalyzesthe addition of a next correct nucleotide to the 3′-OH group of theprimer via a phosphodiester bond, thereby chemically incorporating thenucleotide into the primer. Optionally, the polymerase used in theprovided methods is a processive polymerase. Optionally, the polymeraseused in the provided methods is a distributive polymerase.

As used herein, “biphasic” refers to a two-stage process wherein aprimed template nucleic acid is contacted with a polymerase and a testnucleotide. The first phase of the process involves contacting theprimed template nucleic acid with a polymerase in the presence of asub-saturating level of nucleotide(s), or even in the absence ofnucleotides. The term “sub-saturating,” when used in reference to ligandthat binds to a receptor (e.g., a nucleotide that binds to apolymerase), refers to a concentration of the ligand that is below thatrequired to result in at least 90% of the receptors being bound to theligand at equilibrium. For example, a sub-saturating amount ofnucleotide can yield at least 90%, 95%, 99% or more polymerases beingbound to the nucleotide. The second phase of the process involvescontacting the primed template nucleic acid from the first phase with apolymerase in the presence of a higher concentration of nucleotide(s)than used in the first phase, where the higher concentration issufficient to yield maximal ternary complex formation when a nucleotidein the reaction is the next correct nucleotide.

As used herein, “providing” a template, a primer, or a primed templatenucleic acid refers to the preparation and delivery of one or manynucleic acid polymers, for example to a reaction mixture or reactionchamber.

As used herein, “monitoring” (or sometimes “measuring”), when used inreference to a molecular binding event, refers to a process of detectinga measurable interaction or binding between two molecular species. Forexample, monitoring may involve detecting measurable interactionsbetween a polymerase and primed template nucleic acid (or blocked primedtemplate nucleic acid), typically at various points throughout aprocedure. Monitoring can be intermittent (e.g., periodic) or continuous(e.g., without interruption), and can involve acquisition ofquantitative results. Monitoring can be carried out by detectingmultiple signals over a period of time during a binding event or,alternatively, by detecting signal(s) at a single time point during orafter a binding event.

As used herein, “contacting” refers to the mixing together of reagents(e.g., mixing an immobilized template nucleic acid and either a bufferedsolution that includes a polymerase, or the combination of a polymeraseand a test nucleotide) so that a physical binding reaction or a chemicalreaction may take place.

As used herein, “incorporating” or “chemically incorporating,” when usedin reference to a nucleic acid and nucleotide, refers to the process ofjoining a cognate nucleotide to a nucleic acid primer by formation of aphosphodiester bond.

As used herein, a “binary complex” is a complex between a polymerase anda primed template nucleic acid (or blocked primed template nucleicacid), where the complex does not include a nucleotide molecule such asthe next correct nucleotide.

As used herein, a “ternary complex” is a complex between a polymerase, aprimed template nucleic acid (or blocked primed template nucleic acid),and the next correct nucleotide positioned immediately downstream of theprimer and complementary to the template strand of the primed templatenucleic acid or the blocked primed template nucleic acid. The primedtemplate nucleic acid can include, for example, a primer with a free3′-OH or a blocked primer (e.g., a primer with a chemical modificationon the base or the sugar moiety of the 3′ terminal nucleotide, where themodification precludes enzymatic phosphodiester bond formation).

As used herein, a “catalytic metal ion” refers to a metal ion thatfacilitates phosphodiester bond formation between the 3′-OH of a nucleicacid (e.g., a primer) and the phosphate of an incoming nucleotide by apolymerase. A “divalent catalytic metal cation” is a catalytic metal ionhaving a valence of two. Catalytic metal ions can be present atconcentrations necessary to stabilize formation of a complex between apolymerase, a nucleotide, and a primed template nucleic acid, referredto as non-catalytic concentrations of a metal ion. Catalyticconcentrations of a metal ion refer to the amount of a metal ionsufficient for polymerases to catalyze the reaction between the 3′-OHgroup of a nucleic acid (e.g., a primer) and the phosphate group of anincoming nucleotide.

As used herein, a “non-catalytic metal ion” refers to a metal ion that,when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. Typically, the non-catalytic metal ion is acation. A non-catalytic metal ion may inhibit phosphodiester bondformation by a polymerase, and so may stabilize a ternary complex bypreventing nucleotide incorporation. Non-catalytic metal ions mayinteract with polymerases, for example, via competitive binding comparedto catalytic metal ions. A “divalent non-catalytic metal ion” is anon-catalytic metal ion having a valence of two. Examples of divalentnon-catalytic metal ions include, but are not limited to, Ca²⁺, Zn²⁺,Co²⁺, Ni²⁺, and Sr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalyticmetal ions having a valence of three.

As used herein an “exogenous label” refers to a detectable chemicalmoiety of a sequencing reagent that is not present in a natural analogof the sequencing reagent, such as a non-naturally occurring labelpresent on a synthetic nucleotide analog or a synthetic polymeraseanalog (e.g., a DNA polymerase). While a native dNTP may have acharacteristic limited fluorescence profile, the native dNTP does notinclude any added colorimetric or fluorescent moiety. Conversely, a dATP(2′-deoxyadenosine-5′-triphosphate) molecule modified to include achemical linker and fluorescent moiety attached to the gamma phosphatewould be said to include an exogenous label because the attachedchemical components are not ordinarily a part of the nucleotide. Ofcourse, chemical modifications to add detectable labels to nucleotidebases also would be considered exogenous labels. Likewise, a DNApolymerase modified to include a fluorescent dye (e.g., by attachment toa cysteine residue that is part of the primary sequence of the enzyme)also would be said to include an exogenous label because the label isnot ordinarily a part of the polymerase.

As used herein, “unlabeled” refers to a molecular species free of addedor exogenous label(s) or tag(s). Of course, unlabeled nucleotides willnot include either of an exogenous fluorescent label, or an exogenousRaman scattering tag. A native nucleotide is another example of anunlabeled molecular species. An unlabeled molecular species can excludeone or more of the labels set forth herein or otherwise known in the artrelevant to nucleic acid sequencing or analytical biochemistry.

Sequencing-by-Binding

Described herein are polymerase-based, nucleic acidsequencing-by-binding (SBB) reactions, wherein the polymerase undergoesconformational transitions between open and closed conformations duringdiscrete steps of the reaction. In one step, the polymerase binds to aprimed template nucleic acid to form a binary complex, also referred toherein as the pre-insertion conformation. In a subsequent step, anincoming nucleotide is bound and the polymerase fingers close, forming apre-chemistry conformation including a polymerase, primed templatenucleic acid and nucleotide; wherein the bound nucleotide has not beenincorporated. This step, also referred to herein as the “examination”step, may be followed by a chemical step wherein a phosphodiester bondis formed with concomitant pyrophosphate cleavage from the nucleotide(i.e., nucleotide incorporation). The polymerase, primed templatenucleic acid and newly incorporated nucleotide produce a post-chemistry,pre-translation conformation. As both the pre-chemistry conformation andthe pre-translocation conformation include a polymerase, primed templatenucleic acid and nucleotide, wherein the polymerase is in a closedstate, either conformation may be referred to herein as a closed-complexor a closed ternary complex. In the closed pre-insertion state, divalentcatalytic metal ions, such as Mg²⁺ mediate a rapid chemical reactioninvolving nucleophilic displacement of a pyrophosphate (PPi) by the 3′hydroxyl of the primer. The polymerase returns to an open state upon therelease of PPi, the post-translocation step, and translocation initiatesthe next round of reaction. While a closed-complex can form in theabsence of divalent catalytic metal ions (e.g., Mg²⁺), the polymerase ofthe closed complex is proficient in chemical addition of nucleotide inthe presence of the divalent metal ions. Low or deficient levels ofcatalytic metal ions, such as Mg²⁺, lead to non-covalent (e.g.,physical) sequestration of the next correct nucleotide in aclosed-complex. This closed-complex may be referred to as a stabilizedor trapped closed-complex. In any reaction step described above, thepolymerase configuration and/or interaction with a nucleic acid may bemonitored during an examination step to identify the next correct basein the template nucleic acid sequence. Before or after incorporation,reaction conditions can be changed to disengage the polymerase from theprimed template nucleic acid, and changed again to remove from the localenvironment any reagents that inhibit polymerase binding.

Generally speaking, the SBB procedure includes an “examination” stepthat identifies the next template base, and optionally an“incorporation” step that adds one or more complementary nucleotides tothe 3′-end of the primer component of the primed template nucleic acid.Identity of the next correct nucleotide to be added is determined eitherwithout, or before chemical linkage of that nucleotide to the 3′-end ofthe primer through a covalent bond. The examination step can involveproviding a primed template nucleic acid to be used in the procedure,and contacting the primed template nucleic acid with a polymerase enzyme(e.g., a DNA polymerase) and one or more test nucleotides beinginvestigated as the possible next correct nucleotide. Further, there isa step that involves monitoring or measuring the interaction between thepolymerase and the primed template nucleic acid in the presence of thetest nucleotides. Optionally, the interaction can take place in thepresence of stabilizers, whereby the polymerase-nucleic acid interactionis stabilized in the presence of the next correct nucleotide. Again, theexamination step identifies or determines the identity of the nextcorrect nucleotide without requiring incorporation of that nucleotide.Stated differently, identity of the next correct nucleotide can beestablished without chemical incorporation of the nucleotide into theprimer when one or more cycles of examination is carried out usinglabeled or unlabeled nucleotides.

While methods involving a single template nucleic acid molecule may bedescribed for convenience, these methods are merely exemplary. Thesequencing methods provided herein readily encompass a plurality oftemplate nucleic acids, wherein the plurality of nucleic acids may beclonally amplified copies of a single nucleic acid, or disparate nucleicacids, including combinations, such as populations of disparate nucleicacids that are clonally amplified. Thus, such sequencing methods arefully disclosed herein.

The Examination Step An examination step according to the techniquedescribed herein typically includes the following substeps: (1)providing a primed template nucleic acid (i.e., a template nucleic acidmolecule hybridized with a primer); (2) contacting the primed templatenucleic acid with reaction mixtures that provide two differentconditions (“first” and “second” conditions) with respect to polymeraseand the concentrations of at least one test nucleotide; (3) monitoringthe interaction of the polymerase with the primed template nucleic acidmolecule in the presence of the nucleotide(s) and without chemicalincorporation of any nucleotide into the primed template nucleic acid;and (4) identifying the next base in the template nucleic acid (i.e.,the next correct nucleotide) using the monitored interaction. The primerof the primed template nucleic acid can be an extendible primer. Theprimed template nucleic acid, the polymerase and the test nucleotide arecapable of forming a ternary complex when the base of the testnucleotide is complementary to the next base of the primed templatenucleic acid molecule. Under the second of the conditions in theabove-referenced contacting step, polymerase and test nucleotide areboth present, with the concentration of the test nucleotide beingsufficient to form ternary complexes if the test nucleotide is the nextcorrect nucleotide. Under the first of the conditions in theabove-referenced contacting step, polymerase typically is present atsubstantially the same concentration as in the second condition, but thetest nucleotide is either absent or present at a concentration of 30%,more preferably 20%, or still more preferably 10% or less than theconcentration used in the second condition. The primed template nucleicacid and the polymerase are capable of forming a binary complex when thebase of the test nucleotide is not complementary to the next base of theprimed template nucleic acid molecule. Optionally, the contacting occursunder conditions that favor formation of the ternary complex overformation of the binary complex. The identifying step can includeidentifying the base of the nucleotide that is complementary to the nextbase of the primed template nucleic acid.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, the contacting and monitoring stepscan be repeated one or more times. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a first test nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a second nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a third nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a fourth nucleotide. It is to be understood that eachcontacting step that involves the polymerase and a test nucleotide ispreceded by a contacting step that involves the polymerase and the sametest nucleotide at a lower concentration (e.g., lower by 10-fold ormore) or in the absence of the nucleotide. Thus, the precedingcontacting step may involve contacting with the polymerase in theabsence of the test nucleotide.

In the sequencing methods provided herein, the reaction mixture thatincludes the DNA polymerase and at least one test nucleotide can include1, 2, 3, or 4 types of nucleotide molecules (e.g., either labeled orunlabeled nucleotides). Optionally, the nucleotides are nativenucleotides selected from dATP, dTTP, dCTP, and dGTP. Optionally, thereaction mixture includes one or more triphosphate nucleotides and oneor more diphosphate nucleotides. Optionally, a closed-complex is formedbetween the primed template nucleic acid, the polymerase, and one offour nucleotide molecules included in the reaction mixture.

In a particular example of the provided method, the primed templatenucleic acid is initially contacted with a reaction mixture thatincludes polymerase without added test nucleotide. Thereafter, theprimed template nucleic acid is contacted with a reaction mixture thatincludes polymerase and a first test nucleotide; then with a reactionmixture that includes polymerase and a combination of the first testnucleotide and a second test nucleotide; then with a reaction mixturethat includes polymerase and a combination of the first test nucleotide,the second test nucleotide, and a third test nucleotide; and then with areaction mixture that includes polymerase and a combination of the firsttest nucleotide, the second test nucleotide, the third test nucleotide,and a fourth test nucleotide. Monitoring can take place continuously, orafter each reaction mixture change.

The examination step may be controlled so that nucleotide incorporationis either attenuated or accomplished. If nucleotide incorporation isattenuated during the examination step, then a separate incorporationstep may be performed after determining the identity of the next correctnucleotide. The separate incorporation step may be accomplished withoutthe need for monitoring, as the base has already been identified duringthe examination step. If nucleotide incorporation proceeds duringexamination, subsequent nucleotide incorporation may be attenuated byuse of a stabilizer that traps the polymerase on the nucleic acid afterincorporation. A reversibly terminated nucleotide (i.e., a nucleotideincluding a reversible terminator moiety) may also be used to preventthe addition of subsequent nucleotides. The SBB method allows forcontrolled determination of a template nucleic acid base withoutrequiring the use of labeled nucleotides, as the interaction between thepolymerase and template nucleic acid can be monitored without a label onthe nucleotide. To be clear, however, the use of a labeled nucleotide(e.g., a fluorescent nucleotide) is optional when performing thepresently disclosed procedure to allow for fluorescent detection ofbound nucleotide.

In the sequencing methods provided herein, the test nucleotide (e.g., atleast one test nucleotide) includes a 3′ hydroxyl group, which can be,for example, a free 3′ hydroxyl group. Optionally, the 3′ hydroxyl groupof the test nucleotide molecule is modified to include a 3′ blockingmoiety. The 3′ terminator moiety may be a reversible terminator or maybe an irreversible terminator. Optionally, the reversible terminator ofthe at least one nucleotide molecule is replaced or removed at somepoint after the examination step that employed the test nucleotide thatincluded the reversible terminator.

Contacting Steps

The disclosed method employs two steps for contacting the primedtemplate nucleic acid with a DNA polymerase and one or more testnucleotides. The first step (sometimes referred to herein as the “firstphase,” or “phase 1”) involves contacting the primed template nucleicacid either with the polymerase in the absence of added nucleotide, orwith the polymerase and an amount or concentration of nucleotide that isbelow the amount or concentration needed to achieve maximal formation ofternary closed-complexes when the nucleotide is the next correctnucleotide. For example, a test nucleotide can be used at aconcentration of 30%, more preferably 20%, or still more preferably 10%or less than the concentration used in the second phase. The second step(sometimes referred to herein as the “second phase,” or “phase 2”)involves contacting the primed template nucleic acid with the polymerasein combination with an amount or concentration of nucleotide sufficientto produce maximal ternary complex formation when the test nucleotide isthe next correct nucleotide. This “saturating” level of nucleotide caneasily be determined using no more than reasonable testing. As guidance,the concentration of polymerase used in the first phase can be ≥400 nMin the first phase, and ≤400 nM in the second phase. As furtherguidance, the concentration of dNTP in the first phase can be 0-10 μM,and 20-400 μM in the second phase. While the first and second phases ofthe procedure can use the polymerase at a single concentration, goodresults also have been achieved when the polymerase concentrationsdiffered such that a higher concentration was used in the first phase.Preferably, the polymerase concentration is substantially the same inthe two different contacting steps so that the concentration of the testnucleotide is isolated as the variable in the procedure. Thus, generallyspeaking, the concentration of the test nucleotide during the secondcontacting step will be higher than the concentration of test nucleotidethat may be used in the initial (i.e., first) contacting step.Optionally, the test nucleotide can be omitted entirely from thesolution used for initially contacting primed template nucleic acid. Inthis instance, the first concentration of the test nucleotide will be 0μM. Optionally, the concentration of test nucleotide in the initial(i.e., first) contacting step is so low as to be undetectable byconventional means.

Contacting of the primed template nucleic acid molecule with reactionmixtures that include the polymerase and one or more test nucleotidemolecules can occur under conditions that stabilize formation of theternary complex and/or destabilize formation of the binary complex.Optionally, the reaction mixture includes potassium glutamate.Optionally, the conditions that stabilize formation of the ternarycomplex include contacting the primed template nucleic acid with astabilizing agent. Optionally, the reaction mixture includes astabilizing agent. The stabilizing agent can be one or morenon-catalytic metal ions. Exemplary non-catalytic metal ions includestrontium ion, tin ion, nickel ion, and europium ion. For example, thereaction mixture of the examination step that includes the primedtemplate nucleic acid, the polymerase, and the test nucleotide also mayinclude from 0.01 mM to 30 mM strontium chloride as a stabilizing agent.

In certain embodiments, the primed template nucleic acid is immobilizedto the surface of a solid support. The immobilization may employ eithera covalent or a noncovalent bond between one or the other, or even bothstrands of the primed template nucleic acid and the solid support. Forexample, when the template and primer strands of the primed templatenucleic acid are different molecules, the template strand can beimmobilized, for example via its 5′-end. What is necessary, however, isthat the 3′ terminus of the primer is available for interacting with thepolymerase.

When the primed template nucleic acid is immobilized to a solid support,there are alternatives for how the contacting steps are performed. Forexample, the solid support can be physically transferred betweendifferent vessels (e.g., individual wells of a multiwell plate)containing different reagent solutions. This is convenientlyaccomplished using an automated or robotic instrument. In anotherexample, the primed template nucleic acid is immobilized to a solidsupport inside a flow cell or chamber. In this instance, differentcontacting steps can be executed by controlled flow of different liquidreagents through the chamber, or across the immobilized primed templatenucleic acid.

The Monitoring Step

Monitoring or measuring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule may be accomplished in many different ways. For example,monitoring can include measuring association kinetics for theinteraction between the primed template nucleic acid, the polymerase,and a nucleotide. Monitoring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule can include measuring equilibrium binding constants between thepolymerase and primed template nucleic acid molecule (i.e., equilibriumbinding constants of polymerase to the template nucleic acid in thepresence of a nucleotide). Thus, for example, the monitoring includesmeasuring the equilibrium binding constant of the polymerase to theprimed template nucleic acid in the presence of a nucleotide. Monitoringthe interaction of the polymerase with the primed template nucleic acidmolecule in the presence of a nucleotide molecule includes measuringdissociation kinetics of the polymerase from the primed template nucleicacid in the presence of any one of the four nucleotides. Optionally,monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of a nucleotide molecule includesmeasuring kinetics of the dissociation of the closed-complex (i.e.,dissociation of the primed template nucleic acid, the polymerase, andthe nucleotide). Optionally, the measured association kinetics differdepending on the identity of the nucleotide molecule. Optionally, thepolymerase has a different affinity for each type of nucleotideemployed. Optionally, the polymerase has a different dissociationconstant for each type of nucleotide in each type of closed-complex.Association, equilibrium and dissociation kinetics are known and can bereadily determined by one in the art. See, for example, Markiewicz etal., Nucleic Acids Research 40(16):7975-84 (2012); Xia et al., J. Am.Chem. Soc. 135(1):193-202 (2013); Brown et al., J. Nucleic Acids,Article ID 871939, 11 pages (2010); Washington, et al., Mol. Cell. Biol.24(2):936-43 (2004); Walsh and Beuning, J. Nucleic Acids, Article ID530963, 17 pages (2012); and Roettger, et al., Biochemistry47(37):9718-9727 (2008), which are incorporated by reference herein intheir entireties.

The monitoring step can include monitoring the steady state interactionof the polymerase with the primed template nucleic acid in the presenceof a first nucleotide, without chemical incorporation of the firstnucleotide into the primer of the primed template nucleic acid.Optionally, monitoring includes monitoring the dissociation of thepolymerase from the primed template nucleic acid in the presence of afirst nucleotide, without chemical incorporation of the first nucleotideinto the primer of the primed template nucleic acid. Optionally,monitoring includes monitoring the association of the polymerase withthe primed template nucleic acid in the presence of the firstnucleotide, without chemical incorporation of the first nucleotide intothe primer of the primed template nucleic acid. Again, test nucleotidesin these procedures may be native nucleotides (i.e., unlabeled), labelednucleotides (e.g., fluorescently labeled nucleotides), or nucleotideanalogs (e.g., nucleotides modified to include reversible orirreversible terminator moieties).

In the sequencing methods provided herein, the absence of a catalyticmetal ion in the reaction mixture or the absence of a catalytic metalion in the active site of the polymerase prevents the chemicalincorporation of the nucleotide into the primer of the primed templatenucleic acid. Optionally, the chelation of a catalytic metal ion in thereaction mixtures of the contacting step prevents the chemicalincorporation of the nucleotide into the primer of the primed templatenucleic acid. Optionally, a non-catalytic metal ion acts as a stabilizerfor the ternary closed-complex in the presence of the next correctnucleotide. Optionally, the substitution of a catalytic metal ion in thereaction mixtures of the contacting step with a non-catalytic metal ionprevents the chemical incorporation of the nucleotide molecule to theprimed template nucleic acid. Optionally, the catalytic metal ion ismagnesium. The metal ion mechanisms of polymerases postulates that a lowconcentration of metal ions may be needed to stabilize thepolymerase-nucleotide-DNA binding interaction. See, for instance,Section 27.2.2, Berg J M, Tymoczko J L, Stryer L, Biochemistry 5thEdition, W H Freeman Press, 2002.

Optionally, a low concentration of a catalytic ion in the reactionmixtures of the examination step (i.e., that are used for bindingpolymerase in the presence or absence of a test nucleotide) prevents thechemical incorporation of the test nucleotide into the primer of theprimed template nucleic acid. Optionally, a low concentration of thecatalytic ion (e.g., magnesium ion) is from about 1 μM to about 100 μM.Optionally, a low concentration is from about 0.5 μM to about 5 μM.Optionally, the reaction mixtures of the examination step includecobalt, and the incorporating step includes contacting with anincorporation reaction mixture containing a higher concentration ofcobalt as compared to the concentration of cobalt in the reactionmixtures of the examination step.

The examination step may be controlled, in part, by providing reactionconditions to prevent chemical incorporation of a nucleotide whileallowing monitoring of the interaction between the polymerase and theprimed template nucleic acid, thereby permitting determination of theidentity of the next base of the nucleic acid template strand. Suchreaction conditions may be referred to as “examination reactionconditions.” Optionally, a ternary complex or closed-complex is formedunder examination conditions. Optionally, a stabilized ternary complexor closed-complex is formed under examination conditions or in apre-chemistry conformation.

Optionally, a stabilized closed-complex is in a pre-translocationconformation, wherein the enclosed nucleotide has been incorporated, butthe closed-complex does not allow for the incorporation of a subsequentnucleotide. Optionally, the examination conditions accentuate thedifference in affinity for polymerase to primed template nucleic acidsin the presence of different nucleotides. Optionally, the examinationconditions cause differential affinity of the polymerase to the primedtemplate nucleic acid in the presence of different nucleotides. By wayof example, the examination conditions that cause differential affinityof the polymerase to the primed template nucleic acid in the presence ofdifferent nucleotides include, but are not limited to, high salt andinclusion of potassium glutamate. Concentrations of potassium glutamatethat can be used to alter polymerase affinity for the primed templatenucleic acid include 10 mM to 1.6 M of potassium glutamate, or anyamount in between 10 mM and 1.6 M. Optionally, high salt refers to aconcentration of salt from 50 mM to 1,500 mM salt.

Examination typically involves, in the monitoring step, detectingpolymerase interaction with a template nucleic acid, or with templatenucleic acid and nucleotide in combination. Detection may includeoptical, electrical, thermal, acoustic, chemical and mechanical means.Optionally, monitoring is performed after a buffer change or a washstep, wherein the wash step removes any non-bound reagents (e.g.,unbound polymerases and/or nucleotides) from the region of observation.Optionally, monitoring is performed during a buffer change or a washstep, such that the dissociation kinetics of the polymerase-nucleic acidor polymerase-nucleic acid-nucleotide complexes may be used to determinethe identity of the next base. Optionally, monitoring is performedduring the course of addition of the examination reaction mixture orfirst reaction mixture, such that the association kinetics of thepolymerase to the nucleic acid may be used to determine the identity ofthe next base on the nucleic acid. Optionally, monitoring involvesdistinguishing closed-complexes from binary complexes of polymerase andprimed template nucleic acid. Optionally, monitoring is performed underequilibrium conditions where the affinities measured are equilibriumaffinities. Multiple examination steps including different or similarexamination reagents, may be performed sequentially to ascertain theidentity of the next template base. Multiple examination steps may beutilized in cases where multiple template nucleic acids are beingsequenced simultaneously in one sequencing reaction, wherein differentnucleic acids react differently to the different examination reagents.Optionally, multiple examination steps may improve the accuracy of nextbase determination.

In an exemplary sequencing reaction, the examination step includesformation and/or stabilization of a closed-complex including apolymerase, a primed template nucleic acid, and the next correctnucleotide. Characteristics of the formation and/or release of theclosed-complex are monitored to identify the enclosed nucleotide andtherefore the next base in the template nucleic acid. Closed-complexcharacteristics can be dependent on the sequencing reaction components(e.g., polymerase, primer, template nucleic acid, nucleotide) and/orreaction mixture components and/or conditions. Optionally, theclosed-complex is in a pre-chemistry conformation. Optionally, theclosed-complex is in a pre-translocation conformation. Optionally, theclosed-complex is in a post-translocation conformation.

The examination step involves monitoring the interaction of a polymerasewith a primed template nucleic acid in the presence of a testnucleotide. The formation of a closed-complex may be monitored.Optionally, the absence of formation of a closed-complex is monitored.Optionally, the dissociation of a closed-complex is monitored.Optionally, the incorporation step involves monitoring incorporation ofa nucleotide. Optionally, the incorporation step involves monitoring theabsence of nucleotide incorporation.

Any process of the examination and/or incorporation step may bemonitored. Optionally, a polymerase has an exogenous label or “tag.”Optionally, the detectable tag or label on the polymerase is removable.Optionally, the nucleotides or polymerases have a detectable label,however, the label is not detected during sequencing. Optionally, nocomponent of the sequencing reaction is detectably labeled with anexogenous label.

Monitoring the variation in affinity of a polymerase for a templatenucleic acid in the presence of correct and incorrect nucleotides, underconditions that may or may not allow the incorporation of thenucleotide, may be used to determine the sequence of the nucleic acid.The affinity of a polymerase for a template nucleic acid in the presenceof different nucleotides, including modified or labeled nucleotides, canbe monitored as the off-rate of the polymerase-nucleic acid interactionin the presence of the various nucleotides. The affinities and off-ratesof many standard polymerases to various matched/correct,mismatched/incorrect and modified nucleotides are known in the art.Single molecule imaging of Klenow polymerase reveals that the off-ratefor a template nucleic acid for different nucleotide types, where thenucleotide types are prevented from incorporating, are distinctly andmeasurably different.

Optionally, a nucleotide of a particular type is made available to apolymerase in the presence of a primed template nucleic acid. Thereaction is monitored, wherein, if the nucleotide is a next correctnucleotide, the polymerase may be stabilized to form a closed-complex.If the nucleotide is an incorrect nucleotide, a closed-complex may stillbe formed; however, without the additional assistance of stabilizingagents or reaction conditions (e.g., absence of catalytic ions,polymerase inhibitors, salt), the closed-complex may dissociate. Therate of dissociation is dependent on the affinity of the particularcombination of polymerase, template nucleic acid, and nucleotide, aswell as reaction conditions. Optionally, the affinity is measured as anoff-rate. Optionally, the affinity is different between differentnucleotides for the closed-complex. For example, if the next base in thetemplate nucleic acid downstream of the 3′-end of the primer is G, thepolymerase-nucleic acid affinity, measured as an off-rate, is expectedto be different based on whether dATP, dCTP, dGTP or dTTP are added. Inthis case, dCTP would have the slowest off-rate, with the othernucleotides providing different off-rates for the interaction.Optionally, the off-rate may be different depending on the reactionconditions, for example, the presence of stabilizing agents (e.g.,absence of magnesium or inhibitory compounds) or reaction conditions(e.g., nucleotide modifications or modified polymerases). Once theidentity of the next correct nucleotide is determined, 1, 2, 3, 4 ormore nucleotide types may be introduced simultaneously to the reactionmixture under conditions that specifically target the formation of aclosed-complex. Excess nucleotides may be removed from the reactionmixture and the reaction conditions modulated to incorporate the nextcorrect nucleotide of the closed-complex. This sequencing reactionensures that only one nucleotide is incorporated per sequencing cycle.

The affinity of a polymerase for a template nucleic acid in the presenceof a nucleotide can be measured in a plurality of methods known to oneof skill in the art. Optionally, the affinity is measured as anoff-rate, where the off-rate is measured by monitoring the release ofthe polymerase from the template nucleic acid as the reaction is washedby a wash buffer. Optionally, the affinity is measured as an off-rate,where the off-rate is measured by monitoring the release of thepolymerase from the template nucleic acid under equilibrium bindingconditions, especially equilibrium binding conditions in which thepolymerase binding rates are low or diffusion limited. The polymerasebinding rates may be diffusion limited at sufficiently lowconcentrations of polymerase, wherein if the polymerase falls off fromthe DNA-polymerase complex, it does not load back immediately, allowingfor sufficient time to detect that the polymerase has been released fromthe complex. For a higher affinity interaction, the polymerase isreleased from the nucleic acid slowly, whereas a low affinityinteraction results in the polymerase being released more rapidly. Thespectrum of affinities, in this case, translates to different off-rates,with the off-rates measured under dynamic wash conditions or atequilibrium. The smallest off-rate corresponds to the base complementaryto the added nucleotide, while the other off-rates vary, in a knownfashion, depending on the combination of polymerase and nucleotideselected.

Optionally, the off-rate is measured as an equilibrium signal intensityafter the polymerase and nucleotide are provided in the reactionmixture, wherein the interaction with the lowest off-rate (highestaffinity) nucleotide produces the strongest signal, while theinteractions with other, varying, off-rate nucleotides produce signalsof measurably different intensities. As a non-limiting example, afluorescently labeled polymerase, measured, preferably, under totalinternal reflection (TIRF) conditions, produces different measuredfluorescence intensities depending on the number of polymerase moleculesbound to surface-immobilized nucleic acid molecules in a suitably chosenwindow of time. The intrinsic fluorescence of the polymerase, forinstance, tryptophan fluorescence, may also be utilized. A high off-rateinteraction produces low measured intensities, as the number of boundpolymerase molecules, in the chosen time window is very small, wherein ahigh off-rate indicates that most of the polymerase is unbound from thenucleic acid. Any surface localized measurement scheme may be employedincluding, but not limited to, labeled or fluorescence schemes. Suitablemeasurement schemes that measure affinities under equilibrium conditionsinclude, but are not limited to, bound mass, refractive index, surfacecharge, dielectric constant, and other schemes known in the art.Optionally, a combination of on-rate and off-rate engineering yieldshigher fidelity detection in the proposed schemes. As a non-limitingexample, a uniformly low on-rate, base-dependent, varying off-rateresults in an unbound polymerase remaining unbound for prolongedperiods, allowing enhanced discrimination of the variation in off-rateand measured intensity. The on-rate may be manipulated by lowering theconcentration of the added polymerase, nucleotide, or both polymeraseand nucleotide.

Optionally, the interaction between the polymerase and the nucleic acidis monitored via a detectable tag attached to the polymerase. The tagmay be monitored by detection methods including, but limited to,optical, electrical, thermal, mass, size, charge, vibration, andpressure. The label may be magnetic, fluorescent or charged. Forexternal and internal label schemes, fluorescence anisotropy may be usedto determine the stable binding of a polymerase to a nucleic acid in aclosed-complex.

By way of example, a polymerase is tagged with a fluorophore, whereinclosed-complex formation is monitored as a stable fluorescent signal.The unstable interaction of the polymerase with the template nucleicacid in the presence of an incorrect nucleotide results in a measurablyweaker signal compared to the closed-complex formed in the presence ofthe next correct nucleotide. In certain preferred embodiments, however,the sequencing-by-binding procedure does not rely on detection of anyexogenous label (e.g., a fluorescent label) joined to the polymerase.For example, the polymerase can be a native polymerase.

The Identifying Step

The identity of the next correct base or nucleotide can be determined bymonitoring the presence, formation and/or dissociation of the ternarycomplex or closed-complex. The identity of the next base may bedetermined without chemically incorporating the next correct nucleotideto the 3′-end of the primer. Optionally, the identity of the next baseis determined by monitoring the affinity of the polymerase for theprimed template nucleic acid in the presence of added nucleotides.Optionally, the affinity of the polymerase for the primed templatenucleic acid in the presence of the next correct nucleotide may be usedto determine the next correct base on the template nucleic acid.Optionally, the affinity of the polymerase for the primed templatenucleic acid in the presence of an incorrect nucleotide may be used todetermine the next correct base on the template nucleic acid.

The Incorporation Step

Optionally, the methods provided herein further include an incorporationstep. By way of example, the incorporation step includes incorporating asingle nucleotide (e.g., an unlabeled nucleotide, a reversibleterminator nucleotide, or a detectably labeled nucleotide analog)complementary to the next base of the template nucleic acid into theprimer of the primed template nucleic acid molecule. Optionally, theincorporation step includes contacting the primed template nucleic acidmolecule, polymerase and nucleotide with an incorporation reactionmixture. The incorporation reaction mixture, typically includes acatalytic metal ion.

The provided method may further include preparing the primed templatenucleic acid molecule for a next examination step after theincorporation step. Optionally, the preparing includes subjecting theprimed template nucleic acid or the nucleic acid/polymerase complex toone or more wash steps; a temperature change; a mechanical vibration; apH change; salt or buffer composition changes, an optical stimulation ora combination thereof. Optionally, the wash step includes contacting theprimed template nucleic acid or the primed template nucleicacid/polymerase complex with one or more buffers, detergents, proteindenaturants, proteases, oxidizing agents, reducing agents, or otheragents capable of releasing internal crosslinks within a polymerase orcrosslinks between a polymerase and nucleic acid.

Optionally, the method further includes repeating the examination stepand the incorporation step to sequence a template nucleic acid molecule.The examination step may be repeated one or more times prior toperforming the incorporation step.

Optionally, two consecutive examination steps include reaction mixtureswith different nucleotide molecules (e.g., different nucleotides thatare labeled or unlabeled). Optionally, prior to incorporating the singlenucleotide into the primed template nucleic acid molecule, the firstreaction mixture is replaced with a second reaction mixture including apolymerase and 1, 2, 3, or 4 types of nucleotide molecules (e.g.,different unlabeled nucleotides). Optionally, the nucleotide moleculesare native nucleotides selected from dATP, dTTP, dCTP, and dGTP.

The incorporation reaction may be enabled by an incorporation reactionmixture. Optionally, the incorporation reaction mixture includes adifferent composition of nucleotides than the examination reaction. Forexample, the examination reaction includes one type of nucleotide andthe incorporation reaction includes another type of nucleotide. By wayof another example, the examination reaction includes one type ofnucleotide and the incorporation reaction includes four types ofnucleotides, or vice versa. Optionally, the examination reaction mixtureis altered or replaced by the incorporation reaction mixture.Optionally, the incorporation reaction mixture includes a catalyticmetal ion, potassium chloride, or a combination thereof.

Nucleotides present in the reaction mixture but not sequestered in aclosed-complex may cause multiple nucleotide insertions. Thus, a washstep can be employed prior to the chemical incorporation step to ensureonly the nucleotide sequestered within a trapped closed-complex isavailable for incorporation during the incorporation step. Optionally,free nucleotides may be removed by enzymes such as phosphatases. Thetrapped polymerase complex may be a closed-complex, a stabilizedclosed-complex or ternary complex involving the polymerase, primedtemplate nucleic acid and next correct nucleotide.

Optionally, the nucleotide enclosed within the closed-complex of theexamination step is incorporated into the 3′-end of the template nucleicacid primer during the incorporation step. Optionally, the nucleotideenclosed within the closed-complex of the examination step isincorporated during the examination step, but the closed-complex doesnot allow for the incorporation of a subsequent nucleotide; in thisinstance, the closed-complex is released during an incorporation step,allowing for a subsequent nucleotide to become incorporated.

Optionally, the incorporation step includes replacing a nucleotide fromthe examination step and incorporating another nucleotide into the3′-end of the template nucleic acid primer. The incorporation step canfurther involve releasing a nucleotide from within a closed-complex(e.g., the nucleotide is a modified nucleotide or nucleotide analog) andincorporating a nucleotide of a different kind to the 3′-end of thetemplate nucleic acid primer. Optionally, the released nucleotide isremoved and replaced with an incorporation reaction mixture including anext correct nucleotide.

Suitable reaction conditions for incorporation may involve replacing theexamination reaction mixture with an incorporation reaction mixture.Optionally, nucleotides present in the examination reaction mixture arereplaced with one or more nucleotides in the incorporation reactionmixture. Optionally, the polymerase present during the examination stepis replaced during the incorporation step. Optionally, the polymerasepresent during the examination step is modified during the incorporationstep. Optionally, the one or more nucleotides present during theexamination step are modified during the incorporation step. Thereaction mixture and/or reaction conditions present during theexamination step may be altered by any means during the incorporationstep. These means include, but are not limited to, removing reagents,chelating reagents, diluting reagents, adding reagents, alteringreaction conditions such as conductivity or pH, and any combinationthereof. The reagents in the reaction mixture including any combinationof polymerase, primed template nucleic acid, and nucleotide may bemodified during the examination step and/or incorporation step.

Optionally, the reaction mixture of the incorporation step includescompetitive inhibitors, wherein the competitive inhibitors reduce theoccurrence of multiple incorporations. In certain embodiments, thecompetitive inhibitor is a non-incorporable nucleotide. In certainembodiments, the competitive inhibitor is an aminoglycoside. Thecompetitive inhibitor is capable of replacing either the nucleotide orthe catalytic metal ion in the active site, such that after the firstincorporation the competitive inhibitor occupies the active sitepreventing a second incorporation. In some embodiments, both anincorporable nucleotide and a competitive inhibitor are introduced inthe incorporation step, such that the ratio of the incorporablenucleotide and the inhibitor can be adjusted to ensure incorporation ofa single nucleotide at the 3′-end of the primer.

Optionally, the provided reaction mixtures, including the incorporationreaction mixtures, include at least one unlabeled nucleotide moleculethat is a non-incorporable nucleotide. In other words, the providedreaction mixtures can include one or more unlabeled nucleotide moleculesthat are incapable of incorporation into the primer of the primedtemplate nucleic acid molecule. Nucleotides incapable of incorporationinclude, for example, diphosphate nucleotides. For instance, thenucleotide may contain modifications to the triphosphate group that makethe nucleotide non-incorporable. Examples of non-incorporablenucleotides may be found in U.S. Pat. No. 7,482,120, the disclosure ofwhich is incorporated by reference herein in its entirety. Optionally,the primer may not contain a free hydroxyl group at its 3′-end, therebyrendering the primer incapable of incorporating any nucleotide, and,thus making any nucleotide non-incorporable.

A polymerase inhibitor optionally may be included with the reactionmixtures containing test nucleotides in the examination step to trap thepolymerase on the nucleic acid upon binding the next correct nucleotide.Optionally, the polymerase inhibitor is a pyrophosphate analog.Optionally, the polymerase inhibitor is an allosteric inhibitor.Optionally, the polymerase inhibitor is a DNA or an RNA aptamer.Optionally, the polymerase inhibitor competes with a catalytic-ionbinding site in the polymerase. Optionally, the polymerase inhibitor isa reverse transcriptase inhibitor. The polymerase inhibitor may be anHIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptaseinhibitor. The HIV-1 reverse transcriptase inhibitor may be a(4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one.

In the provided sequencing methods, the next correct nucleotide isidentified before the incorporation step, allowing the incorporationstep to not require labeled reagents and/or monitoring. Thus, in theprovided methods, a nucleotide, optionally, does not contain an attacheddetectable tag or label. Optionally, the nucleotide contains adetectable label, but the label is not detected in the method.Optionally, the correct nucleotide does not contain a detectable label;however, an incorrect or non-complementary nucleotide to the next basecontains a detectable label.

The examination step of the sequencing reaction may be repeated 1, 2, 3,4 or more times prior to the incorporation step. The examination andincorporation steps may be repeated until the desired sequence of thetemplate nucleic acid is obtained.

The formation of the closed-complex or the stabilized closed-complex canbe employed to ensure that only one nucleotide is added to the templatenucleic acid primer per cycle of sequencing, wherein the addednucleotide is sequestered within the closed-complex. The controlledincorporation of a single nucleotide per sequencing cycle enhancessequencing accuracy for nucleic acid regions including homopolymerrepeats.

Reaction Mixtures

Nucleic acid sequencing reaction mixtures, or simply “reactionmixtures,” typically include reagents that are commonly present inpolymerase-based nucleic acid synthesis reactions. Reaction mixturereagents include, but are not limited to, enzymes (e.g., thepolymerase), dNTPs, template nucleic acids, primer nucleic acids, salts,buffers, small molecules, co-factors, metals, and ions. The ions may becatalytic ions, divalent catalytic ions, non-catalytic ions,non-covalent metal ions, or a combination thereof. The reaction mixturecan include salts such as NaCl, KCl, potassium acetate, ammoniumacetate, potassium glutamate, NH₄Cl, or NH₄HSO₄. The reaction mixturecan include a source of ions, such as Mg²⁺ or Mn²⁺ Mg-acetate, Co²⁺ orBa²⁺. The reaction mixture can include tin ions, Ca²⁺, Zn²⁺, Cu²⁺, Co²⁺,Fe²⁺, Ni²⁺, or Eu⁺³. The buffer can include Tris, Tricine, HEPES, MOPS,ACES, MES, phosphate-based buffers, and acetate-based buffers. Thereaction mixture can include chelating agents such as EDTA, EGTA, andthe like. Optionally, the reaction mixture includes cross-linkingreagents. Provided herein are reaction mixtures, optionally, used duringthe examination step, as well as incorporation reaction mixtures usedduring nucleotide incorporation that can include one or more of theaforementioned agents. Reaction mixtures, when used during examination,can be referred to herein as examination reaction mixtures. Optionally,the examination reaction mixture includes a high concentration of salt;a high pH; 1, 2, 3, 4, or more types of unlabeled nucleotides; potassiumglutamate; a chelating agent; a polymerase inhibitor; a catalytic metalion; a non-catalytic metal ion; or any combination thereof. Theexamination reaction mixture can include 10 mM to 1.6 M of potassiumglutamate or any amount in between 10 mM and 1.6 M. Optionally, theincorporation reaction mixture includes a catalytic metal ion; 1, 2, 3,4, or more types of nucleotides (e.g., unlabeled nucleotides); potassiumchloride; a non-catalytic metal ion; or any combination thereof.

Optionally, reaction mixtures in accordance with the disclosedtechniques modulate the formation and stabilization of a closed-complexduring an examination step. For example, the reaction conditions of theexamination step optionally can favor the formation and/or stabilizationof a closed-complex encapsulating a nucleotide, and hinder the formationand/or stabilization of a binary complex. The binary interaction betweenthe polymerase and template nucleic acid may be manipulated bymodulating sequencing reaction parameters such as ionic strength, pH,temperature, or any combination thereof, or by the addition of a binarycomplex destabilizing agent to the reaction. Optionally, high salt(e.g., 50 mM to 1,500 mM) and/or pH changes are utilized to destabilizea binary complex. Optionally, a binary complex may form between apolymerase and a template nucleic acid during the examination orincorporation step of the sequencing reaction, regardless of thepresence of a nucleotide. Optionally, the reaction conditions favor thestabilization of a closed ternary complex and destabilization of abinary complex. By way of example, the pH of the examination reactionmixture can be adjusted from pH 4.0 to pH 10.0 to favor thestabilization of a closed ternary complex and destabilization of abinary complex. Optionally, the pH of the examination reaction mixtureis from pH 4.0 to pH 6.0. Optionally, the pH of the examination reactionmixture is pH 6.0 to pH 10.0.

The provided reaction mixtures and sequencing methods disclosed hereinencourage polymerase interaction with the nucleotides and templatenucleic acid in a manner that reveals the identity of the next basewhile controlling the chemical addition of a nucleotide. Optionally, themethods are performed in the absence of detectably labeled nucleotidesor in the presence of labeled nucleotides wherein the labels are notdetected.

Provided herein are reaction mixtures and methods that facilitateformation and/or stabilization of a closed-complex that includes apolymerase bound to a primed template nucleic acid and a nucleotideenclosed within the polymerase-template nucleic acid complex, underexamination reaction mixture conditions. Examination reaction conditionsmay inhibit or attenuate nucleotide incorporation. Optionally,incorporation of the enclosed nucleotide is inhibited and the complex isstabilized or trapped in a pre-chemistry conformation or a ternarycomplex. Optionally, the enclosed nucleotide is incorporated andsubsequent nucleotide incorporation is inhibited. In this instance, thecomplex is stabilized or trapped in a pre-translocation conformation.For the sequencing reactions provided herein, the closed-complex isstabilized during the examination step, allowing for controllednucleotide incorporation. Optionally, a stabilized closed-complex is acomplex wherein incorporation of an enclosed nucleotide is attenuated,either transiently (e.g., to examine the complex and then incorporatethe nucleotide) or permanently (e.g., for examination only) during anexamination step. Optionally, a stabilized closed-complex allows for theincorporation of the enclosed nucleotide, but does not allow for theincorporation of a subsequent nucleotide. Optionally, the closed-complexis stabilized in order to monitor any polymerase interaction with atemplate nucleic acid in the presence of a nucleotide for identificationof the next base in the template nucleic acid.

Optionally, the enclosed nucleotide has severely reduced or disabledbinding to the template nucleic acid in the closed-complex. Optionally,the enclosed nucleotide is base-paired to the template nucleic acid at anext base. Optionally, the identity of the polymerase, nucleotide,primer, template nucleic acid, or any combination thereof, affects theinteraction between the enclosed nucleotide and the template nucleicacid in the closed-complex.

Optionally, the enclosed nucleotide is bound to the polymerase of theclosed-complex. Optionally, the enclosed nucleotide is weakly associatedwith the polymerase of the closed-complex. Optionally, the identity ofthe polymerase, nucleotide, primer, template nucleic acid, or anycombination thereof, affects the interaction between the enclosednucleotide and the polymerase in the closed-complex. For a givenpolymerase, each nucleotide has a different affinity for the polymerasethan another nucleotide. Optionally, this affinity is dependent, inpart, on the template nucleic acid and/or the primer.

The closed-complex may be transiently formed. Optionally, the enclosednucleotide is a next correct nucleotide. In some methods, the presenceof the next correct nucleotide contributes, in part, to thestabilization of a closed-complex. Optionally, the enclosed nucleotideis not a next correct nucleotide.

Optionally, the examination reaction condition comprises a plurality ofprimed template nucleic acids, polymerases, nucleotides, or anycombination thereof. Optionally, the plurality of nucleotides comprises1, 2, 3, 4, or more types of different nucleotides, for example dATP,dTTP, dGTP, and dCTP. Optionally, the plurality of template nucleicacids is a clonal population of template nucleic acids.

Reaction conditions that may modulate the stability of a closed-complexinclude, but are not limited to, the availability of catalytic metalions, suboptimal or inhibitory metal ions, ionic strength, pH,temperature, polymerase inhibitors, cross-linking reagents, and anycombination thereof. Reaction reagents which may modulate the stabilityof a closed-complex include, but are not limited to, non-incorporablenucleotides, incorrect nucleotides, nucleotide analogs, modifiedpolymerases, template nucleic acids with non-extendible polymerizationinitiation sites, and any combination thereof.

The examination reaction mixture can include other molecules including,but not limited to, enzymes. Optionally, the examination reactionmixture includes any reagents or biomolecules generally present in anucleic acid polymerization reaction. Reaction components may include,but are not limited to, salts, buffers, small molecules, metals, andions. Optionally, properties of the reaction mixture may be manipulated,for example, electrically, magnetically, and/or with vibration.

Nucleotides and Nucleotide Analogs

Nucleotides useful for carrying out the sequencing-by-binding proceduresdescribed herein include native nucleotides, labeled nucleotides (e.g.,nucleotides that include an exogenous fluorescent dye or other label notfound in native nucleotides), and nucleotide analogs (e.g., nucleotideshaving a reversible terminator moiety).

There is flexibility in the nature of the nucleotides that may beemployed in connection with the presently described technique. Anucleotide may include as its nitrogenous base any of: adenine,cytosine, guanine, thymine, or uracil. Optionally, a nucleotide includesinosine, xanthine, hypoxanthine, isocytosine, isoguanine, nitropyrrole(including 3-nitropyrrole) or nitroindole (including 5-nitroindole)base. Useful nucleotides include, but are not limited to, ATP, UTP, CTP,GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP,dUTP, dADP, dTDP, dCDP, dGDP, dUDP, dAMP, dTMP, dCMP, dGMP, and dUMP.Optionally, the phosphate group is modified with a moiety. The moietymay include a detectable label. Optionally, the 3′ OH group of thenucleotide is modified with a moiety. The moiety may be a 3′ reversibleor irreversible terminator. Nucleotides may also contain terminatinginhibitors of DNA polymerase, dideoxynucleotides or 2′, 3′dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddCTP, and ddUTP).

Optionally, a closed-complex of an examination step includes anucleotide analog or modified nucleotide to facilitate stabilization ofthe closed-complex. Optionally, a nucleotide analog includes anitrogenous base, five-carbon sugar, and phosphate group and anycomponent of the nucleotide may be modified and/or replaced. Nucleotideanalogs may be non-incorporable nucleotides. Non-incorporablenucleotides may be modified to become incorporable at any point duringthe sequencing method.

Nucleotide analogs include, but are not limited to, alpha-phosphatemodified nucleotides, alpha-beta nucleotide analogs, beta-phosphatemodified nucleotides, beta-gamma nucleotide analogs, gamma-phosphatemodified nucleotides, caged nucleotides, or ddNTPs. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein in its entirety.

Nucleotide analogs can include terminators that reversibly preventnucleotide incorporation at the 3′-end of the primer. One type ofreversible terminator is a 3′-O-blocked reversible terminator. Here theterminator moiety is linked to the oxygen atom of the 3′-OH end of the5-carbon sugar of a nucleotide. For example, U.S. Pat. No. 7,544,794 andU.S. Pat. No. 8,034,923 (the disclosures of these patents areincorporated by reference) describe reversible terminator dNTPs havingthe 3′-OH group replaced by a 3′-ONH₂ group. This type of reversibleterminator moiety conveniently can be removed (e.g., in a “deblocking”step) using an acetate-buffered solution containing NaNO₂. Another typeof reversible terminator nucleotide is the 2′-modified reversibleterminator described in EP 1 974 057. Yet another type of reversibleterminator is a 3′- unblocked reversible terminator, wherein theterminator moiety is linked to the nitrogenous base of a nucleotide. Forexample, U.S. Pat. No. 8,808,989 (the disclosure of which isincorporated by reference) discloses particular examples ofbase-modified reversible terminator nucleotides that may be used inconnection with the methods described herein. Other reversibleterminators that similarly can be used in connection with the methodsdescribed herein include those described in U.S. Pat. No. 7,956,171,U.S. Pat. No. 8,071,755, and U.S. Pat. No. 9,399,798 (the disclosures ofthese U.S. patents are incorporated by reference). For reviews ofnucleotide analogs having terminators see e.g., Mu, R., et al., “TheHistory and Advances of Reversible Terminators Used in New Generationsof Sequencing Technology,” Genomics, Proteomics & Bioinformatics11(1):34-40 (2013). Optionally, one or more native nucleotides employedduring the examination step is replaced by a second type of nucleotidethat is incorporated during the incorporation step. For example,nucleotides present in the reaction mixture used during an examinationstep may be replaced by nucleotide analogs that include reversibleterminator moieties (e.g., positioned on the base or sugar of thenucleotide molecule).

Optionally, nucleotides are substituted for modified nucleotide analogshaving terminators that irreversibly prevent nucleotide incorporation tothe 3′-end of the primer. Irreversible nucleotide analogs includedideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3 ′-OH group of dNTPs that is essential forpolymerase-mediated synthesis.

Optionally, non-incorporable nucleotides include a blocking moiety thatinhibits or prevents the nucleotide from forming a covalent linkage to asecond nucleotide (3′ OH of a primer) during the incorporation step of anucleic acid polymerization reaction. The blocking moiety can be removedfrom the nucleotide, allowing for nucleotide incorporation.

Optionally, a nucleotide analog present in a closed-complex renders theclosed-complex stable. Optionally, the nucleotide analog isnon-incorporable. Optionally, the nucleotide analog is released and anative nucleotide is incorporated. Optionally, the closed-complex isreleased, the nucleotide analog is modified, and the modified nucleotideanalog is incorporated. Optionally, the closed-complex is released underreaction conditions that modify and/or destabilize the nucleotide analogin the closed-complex.

Optionally, a nucleotide analog present in a closed-complex isincorporated and the closed-complex is stabilized. The closed-complexmay be stabilized by the nucleotide analog, or for example, by anystabilizing methods disclosed herein. Optionally, the nucleotide analogdoes not allow for the incorporation of a subsequent nucleotide. Theclosed-complex can be released, for example, by any methods describedherein, and the nucleotide analog is modified. The modified nucleotideanalog may allow for subsequent incorporation of a nucleotide to its3′-end.

Optionally, a nucleotide analog is present in the reaction mixtureduring the examination step. For example, 1, 2, 3, 4 or more nucleotideanalogs are present in the reaction mixture during the examination step.Optionally, a nucleotide analog is replaced, diluted, or sequesteredduring an incorporation step. Optionally, a nucleotide analog isreplaced with a native nucleotide. The native nucleotide may include anext correct nucleotide. Optionally, a nucleotide analog is modifiedduring an incorporation step. The modified nucleotide analog can besimilar to or the same as a native nucleotide.

Optionally, a nucleotide analog has a different binding affinity for apolymerase than a native nucleotide. Optionally, a nucleotide analog hasa different interaction with a next base than a native nucleotide.Nucleotide analogs and/or non-incorporable nucleotides may base-pairwith a complementary base of a template nucleic acid.

Optionally, a nucleotide analog is a nucleotide, modified or native,fused to a polymerase. Optionally, a plurality of nucleotide analogsincludes fusions to a plurality of polymerases, wherein each nucleotideanalog includes a different polymerase.

A nucleotide can be modified to favor the formation of a closed-complexover the formation of a binary complex. A nucleotide may be selected ormodified to have a high affinity for a polymerase, wherein thepolymerase binds to a nucleotide prior to binding to the templatenucleic acid.

Any nucleotide modification that traps the polymerase in aclosed-complex may be used in the methods disclosed herein. Thenucleotide may be trapped permanently or transiently. Optionally, thenucleotide analog is not the means by which a closed-complex isstabilized. Any closed-complex stabilization method may be combined in areaction utilizing a nucleotide analog.

Optionally, a nucleotide analog that allows for the stabilization of aclosed-complex is combined with reaction conditions that usually releasethe closed-complex. The conditions include, but are not limited to, thepresence of a release reagent (e.g., catalytic metal ion, such asmagnesium or manganese). Optionally, the closed-complex is stabilizedeven in the presence of a catalytic metal ion. Optionally, theclosed-complex is released even in the presence of a nucleotide analog.Optionally, the stabilization of the closed- complex is dependent, inpart, on the concentrations and/or identity of the stabilization reagentand /or release reagents, and any combination thereof. Optionally, thestabilization of a closed-complex using nucleotide analogs is combinedwith additional reaction conditions that function to stabilize aclosed-complex, including, but not limited to, sequestering, removing,reducing, omitting, and/or chelating a catalytic metal ion; the presenceof a polymerase inhibitor, cross-linking agent; and any combinationthereof.

Optionally, one or more nucleotides can be labeled with distinguishingand/or detectable tags or labels; however, such tags or labels are notdetected during examination, identification of the base or incorporationof the base, and are not detected during the sequencing methodsdisclosed herein. The tags may be distinguishable by means of theirdifferences in fluorescence, Raman spectrum, charge, mass, refractiveindex, luminescence, length, or any other measurable property. The tagmay be attached to one or more different positions on the nucleotide, solong as the fidelity of binding to the polymerase-nucleic acid complexis sufficiently maintained to enable identification of the complementarybase on the template nucleic acid correctly. Optionally, the tag isattached to the nucleobase position of the nucleotide. Under suitablereaction conditions, the tagged nucleotides may be enclosed in aclosed-complex with the polymerase and the primed template nucleic acid.Alternatively, a tag is attached to the gamma phosphate position of thenucleotide.

Polymerases

Polymerases useful for carrying out the disclosed sequencing-by-bindingtechnique include naturally occurring polymerases and modified variantsthereof, including, but not limited to, mutants, recombinants, fusions,genetic modifications, chemical modifications, synthetics, and analogs.Naturally occurring polymerases and modified variants thereof are notlimited to polymerases that retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations thereof retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations have special properties that enhance their abilityto sequence DNA, including enhanced binding affinity to nucleic acids,reduced binding affinity to nucleic acids, enhanced catalysis rates,reduced catalysis rates etc. Mutant polymerases include polymeraseswherein one or more amino acids are replaced with other amino acids(naturally or non-naturally occurring), and insertions or deletions ofone or more amino acids. Modified polymerases include polymerases thatcontain an external tag, which can be used to monitor the presence andinteractions of the polymerase. Optionally, intrinsic signals from thepolymerase can be used to monitor their presence and interactions. Thus,the provided methods can include monitoring the interaction of thepolymerase, nucleotide and template nucleic acid through detection of anintrinsic signal from the polymerase. Optionally, the intrinsic signalis a light scattering signal. For example, intrinsic signals includenative fluorescence of certain amino acids such as tryptophan, whereinchanges in intrinsic signals from the polymerase may indicate theformation of a closed-complex. Thus, in the provided methods, thepolymerase is an unlabeled polymerase and monitoring is performed in theabsence of a detectable label associated with the polymerase. Somemodified polymerases or naturally occurring polymerases, under specificreaction conditions, may incorporate only single nucleotides and mayremain bound to the primer-template after the incorporation of thesingle nucleotide. Optionally, the thumb and finger domains of thepolymerase may form transient or covalent crosslinks due to theirphysical proximity in the closed form of the polymerase. The crosslinksmay be formed, for example by native or engineered cysteines at suitablepositions on the thumb and finger domains.

The term polymerase and its variants, as used herein, also refers tofusion proteins including at least two portions linked to each other,for example, where one portion includes a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand is linked toanother portion that includes a second moiety, such as, a reporterenzyme or a processivity-modifying domain. For example, T7 DNApolymerase includes a nucleic acid polymerizing domain and a thioredoxinbinding domain, wherein thioredoxin binding enhances the processivity ofthe polymerase. Absent the thioredoxin binding, T7 DNA polymerase is adistributive polymerase with processivity of only one to a few bases.Although DNA polymerases differ in detail, they have a similar overallshape of a hand with specific regions referred to as the fingers, thepalm, and the thumb; and a similar overall structural transition,including the movement of the thumb and/or finger domains, during thesynthesis of nucleic acids.

DNA polymerases include, but are not limited to, bacterial DNApolymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viralDNA polymerases and phage DNA polymerases. Bacterial DNA polymerasesinclude E. coli DNA polymerases I, II and III, IV and V, the Klenowfragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNApolymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and κ, as well as the Revlpolymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cp1 DNA polymerase, Cp7 DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other DNA polymerasesinclude thermostable and/or thermophilic DNA polymerases such as DNApolymerases isolated from Thermus aquaticus (Taq) DNA polymerase,Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu(Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcusfuriosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcuslitoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D polymerase,Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus(Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, PfxDNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase,Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophiliumDNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcussp. go N-7 DNA polymerase; Pyrodictium occultum DNA polymerase;Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicumDNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcusstrain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNApolymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicumDNA polymerase; Thermococcus fumicolans DNA polymerase; Aeropyrum pernixDNA polymerase; and the heterodimeric DNA polymerase DP1/DP2. Engineeredand modified polymerases also are useful in connection with thedisclosed techniques. For example, modified versions of the extremelythermophilic marine archaea Thermococcus species 9° N (e.g., TherminatorDNA polymerase from New England BioLabs Inc.; Ipswich, Mass.) can beused.

RNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11polymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNApolymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymeraseV; and Archaea RNA polymerases.

Reverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andtelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

Optionally, a polymerase is tagged with a chemiluminescent tag, whereinclosed-complex formation is monitored as a stable luminescence signal inthe presence of the appropriate luminescence triggers. The unstableinteraction of the polymerase with the template nucleic acid in thepresence of an incorrect nucleotide results in a measurably weakersignal compared to the closed-complex formed in the presence of the nextcorrect nucleotide. Additionally, a wash step prior to triggeringluminescence could remove all polymerase molecules not bound in a stableclosed-complex.

Optionally, a polymerase is tagged with an optical scattering tag,wherein closed-complex formation is monitored as a stable opticalscattering signal. The unstable interaction of the polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, the polymerase is tagged with a plasmonic nanoparticle tag,wherein the closed-complex formation is monitored as a shift inplasmonic resonance that is different from the plasmonic resonance inthe absence of the closed-complex or the presence of a closed-complexincluding an incorrect nucleotide. The change in plasmon resonance maybe due to the change in local dielectric environment in theclosed-complex, or it may be due to the synchronous aggregation of theplasmonic nanoparticles on a cluster of clonally amplified nucleic acidmolecules or another means that affects the plasmons differently in theclosed-complex configuration.

Optionally, the polymerase is tagged with a Raman scattering tag,wherein the closed-complex formation is monitored as a stable Ramanscattering signal. The unstable interaction of polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, a next correct nucleotide is identified by a tag on apolymerase selected or modified to have a high affinity for nucleotides,wherein the polymerase binds to a nucleotide prior to binding to thetemplate nucleic acid. For example, the DNA polymerase X from theAfrican Swine Fever virus has an altered order of substrate binding,where the polymerase first binds to a nucleotide, then binds to thetemplate nucleic acid. Optionally, a polymerase is incubated with eachtype of nucleotide in separate compartments, where each compartmentcontains a different type of nucleotide and where the polymerase islabeled differently with a tag depending on the nucleotide with which itis incubated. In these conditions, unlabeled nucleotides are bound todifferently labeled polymerases. The polymerases may be the same kind ofpolymerase bound to each nucleotide type or different polymerases boundto each nucleotide type. The differentially tagged polymerase-nucleotidecomplexes may be added simultaneously to any step of the sequencingreaction. Each polymerase-nucleotide complex binds to a template nucleicacid whose next base is complementary to the nucleotide in thepolymerase-nucleotide complex. The next correct nucleotide is identifiedby the tag on the polymerase carrying the nucleotide. The interrogationof the next template base by the labeled polymerase-nucleotide complexmay be performed under non-incorporating and/or examination conditions,where once the identity of the next template base is determined, thecomplex is destabilized and removed, sequestered, and/or diluted and aseparate incorporation step is performed in a manner ensuring that onlyone nucleotide is incorporated.

A common method of introducing a detectable tag on a polymeraseoptionally involves chemical conjugation to amines or cysteines presentin the non-active regions of the polymerase. Such conjugation methodsare well known in the art. As non-limiting examples,n-hydroxysuccinimide esters (NHS esters) are commonly employed to labelamine groups that may be found on an enzyme. Cysteines readily reactwith thiols or maleimide groups, while carboxyl groups may be reactedwith amines by activating them with EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).Optionally, N-hydroxysuccinimide (NHS) chemistry is employed at pHranges where only the N-terminal amines are reactive (for instance, pH7), such that only a single tag is added per polymerase.

Optionally, the tag attached to the polymerase is a charge tag, suchthat the formation of stable closed-complex can be detected byelectrical means by measuring changes in local charge density around thetemplate nucleic acids. Methods for detecting electrical charges arewell known in the art, including methods such as field-effecttransistors, dielectric spectroscopy, impedance measurements, and pHmeasurements, among others. Field-effect transistors include, but arenot limited to, ion-sensitive field-effect transistors (ISFET), charge-modulated field-effect transistors, insulated-gate field-effecttransistors, metal oxide semiconductor field-effect transistors andfield-effect transistors fabricated using semiconducting single wallcarbon nanotubes.

Optionally, a charge tag is a peptide tag having an isoelectric pointbelow about 4 or above about 10. Optionally, a polymerase including apeptide tag has a total isoelectric point below about 5 or above about9. A charge tag may be any moiety which is positively or negativelycharged. The charge tag may include additional moieties including massand/or labels such as dyes. Optionally, the charge tag possesses apositive or negative charge only under certain reaction conditions suchas changes in pH.

A polymerase may be labeled with a fluorophore and/or quencher.Optionally, a nucleic acid is labeled with a fluorophore and/orquencher. Optionally, one or more nucleotides are labeled with afluorophore and/or quencher. Exemplary fluorophores include, but are notlimited to, fluorescent nanocrystals; quantum dots; d-Rhodamine acceptordyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA],dichloro[ROX] or the like; fluorescein donor dye including fluorescein,6-FAM, or the like; Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes,Atto dyes such as atto 647N which forms a FRET pair with Cy3B and thelike. Fluorophores include, but are not limited to, MDCC(7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), TET,HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705 and LC red 640.Fluorophores and methods for their use including attachment topolymerases and other molecules are described in The Molecular Probes®Handbook (Life Technologies; Carlsbad Calif) and Fluorophores Guide(Promega; Madison, Wis.), which are incorporated herein by reference intheir entireties. Exemplary quenches include, but are not limited to,ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qx1 quencher, Iowa BlackRQ, and IRDye QC-1.

Optionally, a conformationally sensitive dye may be attached close tothe active site of the polymerase without affecting the polymerizationability or fidelity of the polymerase; wherein a change in conformation,or a change in polar environment due to the formation of aclosed-complex is reflected as a change in fluorescence or absorbanceproperties of the dye. Common fluorophores such as Cy3 and fluoresceinare known to have strong solvatochromatic response to polymerase bindingand closed-complex formation, to the extent that the formation ofclosed-complex can be distinguished clearly from the binarypolymerase-nucleic acid complex. Optionally, the closed-complex can bedistinguished from binary complexes based on differences in fluorescenceor absorbance signals from a conformationally sensitive dye. Optionally,a solvatochromatic dye may be employed to monitor conformationaltransitions; wherein the change in local polar environment induced bythe conformational change can be used as the reporter signal.Solvatochromatic dyes include, but are not limited to, Reichart's dye,IR44, merocyanine dyes (e.g., merocyanine 540), 4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-l-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, indigoid dyes, as exemplified by indigo, and others as well asmixtures thereof. Methods to introduce dyes or fluorophores to specificsites of a polymerase are well known in the art. As a non-limitingexample, a procedure for site specific labeling of a T7 DNA polymerasewith a dye is provided by Tsai et al., in “Site-Specific Labeling of T7DNA Polymerase with a Conformationally Sensitive Fluorophore and Its Usein Detecting Single-Nucleotide Polymorphisms,” Analytical Biochemistry384: 136-144 (2009), which is incorporated by reference herein in itsentirety.

Optionally, a polymerase is tagged with a fluorophore at a position thatcould sense closed-complex formation without interfering with thereaction. The polymerase may be a native or modified polymerase.Modified polymerases include those with one or more amino acidmutations, additions, and/or deletions. Optionally, one or more, but notall, cysteine amino acids are mutated to another amino acid, such asalanine. In this case, the remaining one or more cysteines are used forsite-specific conjugation to a fluorophore. Alternatively, one or moreamino acids are mutated to a reactive amino acid suitable forfluorophore conjugation, such as cysteines or amino acids includingprimary amines.

Optionally, binding between a polymerase and a template nucleic acid inthe presence of a correct nucleotide may induce a decrease influorescence, whereas binding with an incorrect nucleotide causes anincrease in fluorescence. Binding between a polymerase and a templatenucleic acid in the presence of a correct nucleotide may induce anincrease in fluorescence, whereas binding with an incorrect nucleotidecauses a decrease in fluorescence. The fluorescent signals may be usedto monitor the kinetics of a nucleotide-induced conformational changeand identify the next base in the template nucleic acid sequence.

Optionally, the polymerase/nucleic-acid interaction may be monitored byscattering signal originating from the polymerase or tags attached tothe polymerase, for instance, nanoparticle tags.

Conditions for Forming and Manipulating Closed-Complexes

As used herein, a closed-complex can be a ternary complex that includesa polymerase, primed template nucleic acid, and nucleotide. Theclosed-complex may be in a pre-chemistry conformation, wherein anucleotide is sequestered but not incorporated. The closed-complex mayalternatively be in a pre-translocation conformation, wherein anucleotide is incorporated by formation of a phosphodiester bond withthe 3′-end of the primer in the primed template nucleic acid. Theclosed-complex may be formed in the absence of catalytic metal ions ordeficient levels of catalytic metal ions, thereby physicallysequestering the next correct nucleotide within the polymerase activesite without chemical incorporation. Optionally, the sequesterednucleotide may be a non-incorporable nucleotide. The closed-complex maybe formed in the presence of catalytic metal ions, where theclosed-complex includes a nucleotide analog which is incorporated, but aPPi is not capable of release. In this instance, the closed-complex isstabilized in a pre-translocation conformation. Optionally, apre-translocation conformation is stabilized by chemically cross-linkingthe polymerase. Optionally, the closed-complex may be stabilized byexternal means. In some instances, the closed-complex may be stabilizedby allosteric binding of small molecules, or macromolecules such asantibodies or aptamers. Optionally, closed-complex may be stabilized bypyrophosphate analogs that bind close to the active site with highaffinity, preventing translocation of the polymerase.

As used herein, a stabilized closed-complex or stabilized ternarycomplex refers to a polymerase trapped at the polymerization initiationsite (3′-end of the primer) of the primed template nucleic acid by oneor a combinations of means, including but not limited to, crosslinkingthe thumb and finger domains in the closed conformation, binding of anallosteric inhibitor that prevents return of the polymerase to an openconformation, binding of pyrophosphate analogs that trap polymerase inthe pre-translocation step, absence of catalytic metal ions in theactive site of the polymerase, and addition of a metal ions such asnickel, tin and Sr²⁺ as substitutes for a catalytic metal ion. As such,the polymerase may be trapped at the polymerization initiation site evenafter the incorporation of a nucleotide. Therefore, the polymerase maybe trapped in the pre-chemistry conformation, pre-translocation step,post-translocation step or any intermediate step thereof. Thus, allowingfor sufficient examination and identification of the next correctnucleotide or base.

As described herein, a polymerase-based, sequencing-by-binding reactiongenerally involves providing a primed template nucleic acid with apolymerase and one or more types of nucleotides, wherein the nucleotidesmay or may not be complementary to the next base of the primed templatenucleic acid, and examining the interaction of the polymerase with theprimed template nucleic acid under conditions wherein either chemicalincorporation of a nucleotide into the primed template nucleic acid isdisabled or severely inhibited in the pre-chemistry conformation or oneor more complementary nucleotide incorporation occurs at the 3′-end ofthe primer. Optionally, wherein the pre- chemistry conformation isstabilized prior to nucleotide incorporation, preferably usingstabilizers, a separate incorporation step may follow the examinationstep to incorporate a single nucleotide to the 3′-end of the primer.Optionally, where a single nucleotide incorporation occurs, thepre-translocation conformation may be stabilized to facilitateexamination and/or prevent subsequent nucleotide incorporation.

As indicated above, the presently described methods for sequencing anucleic acid include an examination step. The examination step involvesbinding a polymerase to the polymerization initiation site of a primedtemplate nucleic acid in a reaction mixture including one or morenucleotides, and monitoring the interaction. Optionally, a nucleotide issequestered within the polymerase-primed template nucleic acid complexto form a closed-complex, under conditions in which incorporation of theenclosed nucleotide by the polymerase is attenuated or inhibited.Optionally a stabilizer is added to stabilize the ternary complex in thepresence of the next correct nucleotide. This closed-complex is in astabilized or polymerase-trapped pre-chemistry conformation. Aclosed-complex allows for the incorporation of the enclosed nucleotidebut does not allow for the incorporation of a subsequent nucleotide.This closed-complex is in a stabilized or trapped pre-translocationconformation. Optionally, the polymerase is trapped at thepolymerization site in its closed-complex by one or a combination ofmeans including, but not limited to, crosslinking of the polymerasedomains, crosslinking of the polymerase to the nucleic acid, allostericinhibition by small molecules, uncompetitive inhibitors, competitiveinhibitors, non-competitive inhibitors, and denaturation; wherein theformation of the trapped closed-complex provides information about theidentity of the next base on the nucleic acid template.

Optionally, a closed-complex is released from its trapped or stabilizedconformation, which may allow for nucleotide incorporation to the 3′-endof the template nucleic acid primer. The closed-complex can bedestabilized and/or released by modulating the composition of thereaction conditions. In addition, the closed-complex can be destabilizedby electrical, magnetic, and/or mechanical means. Mechanical meansinclude mechanical agitation, for example, by using ultrasoundagitation. Mechanical vibration destabilizes the closed-complex andsuppresses binding of the polymerase to the DNA. Thus, rather than awash step where the examination reaction mixture is replaced with anincorporation mixture, mechanical agitation may be used to remove thepolymerase from the template nucleic acid, enabling cycling throughsuccessive incorporation steps with a single nucleotide addition perstep.

Any combination of closed-complex stabilization or closed-complexrelease reaction conditions and/or methods may be combined. For example,a polymerase inhibitor that stabilizes a closed-complex may be presentin the examination reaction with a catalytic ion, which functions torelease the closed-complex. In the aforementioned example, theclosed-complex may be stabilized or released, depending on thepolymerase inhibitor properties and concentration, the concentration ofthe catalytic metal ion, other reagents and/or conditions of thereaction mixture, and any combination thereof.

The closed-complex can be stabilized under reaction conditions wherecovalent attachment of a nucleotide to the 3′-end of the primer in theprimed template nucleic acid is attenuated. Optionally, theclosed-complex is in a pre-chemistry conformation or ternary complex.Optionally, the closed-complex is in a pre-translocation conformation.The formation of this closed-complex can be initiated and/or stabilizedby modulating the availability of a catalytic metal ion that permitsclosed-complex release and/or chemical incorporation of a nucleotide tothe primer in the reaction mixture. Exemplary metal ions include, butare not limited to, magnesium, manganese, cobalt, and barium. Catalyticions may be any formulation, for example, salts such as MgCl₂,Mg(CH₃CO₂)₂, and MnCl₂.

The selection and/or concentration of the catalytic metal ion may bebased on the polymerase and/or nucleotides in the sequencing reaction.For example, the HIV reverse transcriptase utilizes magnesium fornucleotide incorporation (N Kaushik, Biochemistry 35:11536-11546 (1996),and H P Patel, Biochemistry 34:5351-5363 (1995), which are incorporatedby reference herein in their entireties). The rate of closed-complexformation using magnesium versus manganese can be different depending onthe polymerase and the identity of the nucleotide. Thus, the stabilityof the closed-complex may differ depending on catalytic metal ion,polymerase, and/or nucleotide identity. Further, the concentration ofcatalytic ion necessary for closed-complex stabilization may varydepending on the catalytic metal ion, polymerase, and/or nucleotideidentity and can be readily determined using the guidance providedherein. For example, nucleotide incorporation may occur at highcatalytic ion concentrations of one metal ion but does not occur at lowconcentrations of the same metal ion, or vice versa. Therefore,modifying metal ion identity, metal ion concentration, polymeraseidentity, and/or nucleotide identity allows for controlled examinationreaction conditions.

The closed-complex may be formed and/or stabilized by sequestering,removing, reducing, omitting, and/or chelating a catalytic metal ionduring the examination step of the sequencing reaction so thatclosed-complex release and/or chemical incorporation does not occur.Chelation includes any procedure that renders the catalytic metal ionunavailable for nucleotide incorporation, including using EDTA and/orEGTA. A reduction includes diluting the concentration of a catalyticmetal ion in the reaction mixture. The reaction mixture can be dilutedor replaced with a solution including a non-catalytic ion, which permitsclosed-complex formation, but inhibits nucleotide incorporation.Non-catalytic ions include, but are not limited to, calcium, strontium,scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper,zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium.Optionally, Ni²⁺ is provided in an examination reaction to facilitateclosed-complex formation. Optionally, Sr²⁺ is provided in an examinationreaction to facilitate closed-complex formation. Optionally, anon-catalytic metal ion and a catalytic metal ion are both present inthe reaction mixture, wherein one ion is present in a higher effectiveconcentration than the other. In the provided methods, a non-catalyticion such as cobalt can become catalytic (i.e., facilitate nucleotideincorporation) at high concentrations. Thus, optionally, a lowconcentration of a non-catalytic metal ion is used to facilitate ternarycomplex formation and a higher concentration of the non-catalytic metalion is used to facilitate incorporation.

Non-catalytic ions may be added to a reaction mixture under examinationconditions. The reaction may already include nucleotides. Optionally,non-catalytic ions are complexed to one or more nucleotides andcomplexed nucleotides are added to the reaction mixture. Non-catalyticions can complex to nucleotides by mixing nucleotides with non-catalyticions at elevated temperatures (about 80° C.). For example, a chromiumnucleotide complex may be added to a mixture to facilitateclosed-complex formation and stabilization. Optionally, a chromiumnucleotide complex is a chromium monodentate, bidentate, or tridentatecomplex. Optionally, a chromium nucleotide complex is an a-monodentate,or β- γ-bidentate nucleotide.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingSr²⁺, wherein Sr²⁺ promotes the formation of the closed-complex. Thepresence of Sr²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Sr²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Sr²⁺ ispresent as 10 mM SrCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash step removesunbound nucleotides, and Mg²⁺ is added to the reaction to inducepyrophosphate (PPi) cleavage and nucleotide incorporation. Optionally,the wash step includes Sr²⁺ to maintain the stability of the ternarycomplex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read-length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingNi²⁺, wherein Ni²⁺ promotes the formation of the closed-complex. Thepresence of Ni²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Ni²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Ni²⁺ ispresent as 10 mM NiCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Mg²⁺ is added to the reaction toinduce pyrophosphate (PPi) cleavage and nucleotide incorporation.Optionally, the wash buffer includes Ni²⁺ to maintain the stability ofthe ternary complex, preventing the dissociation of the ternary complex.The reaction may be repeated until a desired sequence read length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingnon-catalytic concentrations of Co²⁺, wherein Co²⁺ promotes theformation of the closed-complex. The presence of non-catalyticconcentrations of Co²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Co²⁺ ion may be presentat concentrations from about 0.01 mM to about 0.5 mM. Optionally, Co²⁺is present as 0.5 mM CoCl₂. The formation of the closed-complex ismonitored under examination conditions to identify the next base in thetemplate nucleic acid of the closed-complex. The affinity of thepolymerase (e.g., Klenow fragment of E. coli DNA polymerase I, Bst) foreach of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Co²⁺can be different. Therefore, examination can involve measuring thebinding affinities of polymerase-template nucleic acids to dNTPs;wherein binding affinity is indicative of the next base in the templatenucleic acid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Co²⁺ at a catalyticconcentration is added to the reaction to induce pyrophosphate (PPi)cleavage and nucleotide incorporation. Optionally, the wash bufferincludes non-catalytic amounts of Co²⁺ to maintain the stability of theternary complex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read length isobtained.

Optionally, a catalytic metal ion may facilitate the formation of aclosed-complex without subsequent nucleotide incorporation andclosed-complex release. Optionally, a concentration of 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 μM Mg²⁺ in a reaction mixture can induceconformational change of a polymerase to form a closed-complex withoutsubsequent nucleotide incorporation, PPi and closed-complex release.Optionally, the concentration of Mg²⁺ is from about 0.5 μM to about 10μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 4 μM,from about 0.5 μM to about 3 μM, from about μM to about 5 μM, from about1 μM to about 4 μM, and from about 1 μM to about 3 μM.

Optionally, the concentration of available catalytic metal ion in thesequencing reaction which is necessary to allow nucleotide incorporationis from about 0.001 mM to about 10 mM, from about 0.01 mM to about 5 mM,from about 0.01 mM to about 3 mM, from about 0.01 mM to about 2 mM, fromabout 0.01 mM to about 1 mM, from about 0.05 mM to about 10 mM, fromabout 0.05 mM to about 5 mM, from about 0.05 mM to about 3 mM, fromabout 0.05 to about 2 mM, or from about 0.05 mM to about 1 mM.Optionally, the concentration of catalytic metal ion is from 5 mM to 50mM. Optionally, the concentration of catalytic metal ion is from 5 mM to15 mM, or about 10 mM.

A non-catalytic ion may be added to the reaction mixture at any stageincluding before, during, or after any of the following reaction steps:providing a primed template nucleic acid, providing a polymerase,formation of a binary complex, providing a nucleotide, formation of apre-chemistry closed-complex, nucleotide incorporation, formation of apre-translocation closed-complex, and formation of a post-translocationconformation. The non-catalytic ion may be added to the reaction mixtureduring wash steps. The non-catalytic ion may be present through thereaction in the reaction mixture. For example, a catalytic ion is addedto the reaction mixture at concentrations which dilute the non-catalyticmetal ion, allowing for nucleotide incorporation.

The ability of catalytic and non-catalytic ions to modulate nucleotideincorporation may depend on conditions in the reaction mixtureincluding, but not limited to, pH, ionic strength, chelating agents,chemical cross-linking, modified polymerases, non-incorporablenucleotides, mechanical or vibration energy, and electric fields.

Optionally, the concentration of non-catalytic metal ion in thesequencing reaction necessary to allow for closed-complex formationwithout nucleotide incorporation is from about 0.1 mM to about 50 mM,from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, fromabout 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, fromabout 0.1 mM to about 5 mM, from about 0.1 to about 1 mM, from about 1mM to about 50 mM, from about 1 to about 40 mM, from about 1 mM to about30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM,from about 1 mM to about 5 mM, from about 2 mM to about 30 mM, fromabout 2 mM to about 20 mM, from about 2 mM to about 10 mM, or anyconcentration within these ranges.

A closed-complex may be formed and/or stabilized by the addition of apolymerase inhibitor to the examination reaction mixture. Inhibitormolecules phosphonoacetate (phosphonoacetic acid) and phosphonoformate(phosphonoformic acid, common name Foscarnet), Suramin, Aminoglycosides,INDOPY-1 and Tagetitoxin are non-limiting examples of uncompetitive ornoncompetitive inhibitors of polymerase activity. The binding of theinhibitor molecule, near the active site of the enzyme, traps thepolymerase in either a pre-translocation or post-translocation step ofthe nucleotide incorporation cycle, stabilizing the polymerase in itsclosed-complex conformation before or after the incorporation of anucleotide, and forcing the polymerase to be bound to the templatenucleic acid until the inhibitor molecules are not available in thereaction mixture by removal, dilution or chelation.

Thus, provided is a method for sequencing a template nucleic acidmolecule including an examination step including providing a templatenucleic acid molecule primed with a primer; contacting the primedtemplate nucleic acid molecule with a first reaction mixture including apolymerase, a polymerase inhibitor and at least one unlabeled nucleotidemolecule; monitoring the interaction of the polymerase with the primedtemplate nucleic acid molecule in the presence of the unlabelednucleotide molecule without incorporation of the nucleotide into theprimer of the primed template nucleic acid molecule; and identifying thenucleotide that is complementary to the next base of the primed templatenucleic acid molecule by the monitored interaction. The polymeraseinhibitor prevents the incorporation of the unlabeled nucleotidemolecule into the primer of the primer template nucleic acid.Optionally, the inhibitor is a non-competitive inhibitor, an allostericinhibitor, or an uncompetitive allosteric inhibitor. Optionally, thepolymerase inhibitor competes with a catalytic ion binding site in thepolymerase.

Detection Platforms: Instrumentation for Detecting the Closed-Complex

The interaction between the polymerase and the template nucleic acid inthe presence of nucleotides can be monitored without the use of anexogenous label. For example, the sequencing reaction may be monitoredby detecting the change in refractive index, charge detection, Ramanscattering detection, ellipsometry detection, pH detection, sizedetection, mass detection, surface plasmon resonance, guided moderesonance, nanopore optical interferometry, whispering gallery moderesonance, nanoparticle scattering, photonic crystal, quartz crystalmicrobalance, bio-layer interferometry, vibrational detection, pressuredetection and other label-free detection schemes that detect the addedmass or refractive index due to polymerase binding in a closed-complexwith a template nucleic acid.

Optionally, detecting a change in refractive index is accomplished byone or a combination of means, including, but not limited to, surfaceplasmon resonance sensing, localized plasmon resonance sensing,plasmon-photon coupling sensing, transmission sensing throughsub-wavelength nanoholes (enhanced optical transmission), photoniccrystal sensing, interferometry sensing, refraction sensing, guided moderesonance sensing, ring resonator sensing, or ellipsometry sensing.Optionally, nucleic acid molecules may be localized to a surface,wherein the interaction of polymerase with nucleic acids in the presenceof various nucleotides may be measured as a change in the localrefractive index.

Optionally, the template nucleic acid is tethered to or localizedappropriately on or near a surface, such that the interaction ofpolymerase and template nucleic acid in the presence of nucleotideschanges the light transmitted across or reflected from the surface. Thesurface may contain nanostructures. Optionally, the surface is capableof sustaining plasmons or plasmon resonance. Optionally, the surface isa photonic substrate, not limited to a resonant cavity, resonant ring orphotonic crystal slab. Optionally, the surface is a guided moderesonance sensor. Optionally, the nucleic acid is tethered to, orlocalized appropriately on or near a nanohole array, a nanoparticle or amicroparticle, such that the interaction of polymerase and templatenucleic acid in the presence of nucleotides changes the absorbance,scattering, reflection or resonance of the light interacting with themicroparticle or nanoparticle.

Optionally, a nanohole array on a gold surface is used as a refractiveindex sensor. The template nucleic acid may be attached to a metalsurface by standard thiol chemistry, incorporating the thiol group onone of the primers used in a PCR reaction to amplify the DNA. When thedimensions of the nanohole array are appropriately tuned to the incidentlight, binding of the polymerase to the template nucleic acid in thepresence of nucleotides can be monitored as a change in lighttransmitted across the nanoholes. For both the labeled and label-freeschemes, simple and straightforward measurement of equilibrium signalintensity may reveal the formation of a stable closed-complex.

Optionally, nucleic acid molecules are localized to a surface capable ofsustaining surface plasmons, wherein the change in refractive indexcaused by the polymerase interaction with localized nucleic acids may bemonitored through the change in the properties of the surface plasmons,wherein further, said properties of surface plasmons may include surfaceplasmon resonance. Surface plasmons, localized surface plasmons (LSP),or surface plasmon polaritons (SPP), arise from the coupling ofelectromagnetic waves to plasma oscillations of surface charges. LSPsare confined to nanoparticle surfaces, while SPPs and are confined tohigh electron density surfaces, at the interface between high electronmobility surfaces and dielectric media. Surface plasmons may propagatealong the direction of the interface, whereas they penetrate into thedielectric medium only in an evanescent fashion. Surface plasmonresonance conditions are established when the frequency of incidentelectromagnetic radiation matches the natural frequency of oscillationof the surface electrons. Changes in dielectric properties at theinterface, for instance due to binding or molecular crowding, affectsthe oscillation of surface electrons, thereby altering the surfaceplasmon resonance wavelength. Surfaces capable of surface plasmonresonance include, in a non-limiting manner, nanoparticles, clusters andaggregates of nanoparticles, continuous planar surfaces, nanostructuredsurfaces, and microstructured surfaces. Materials such as gold, silver,aluminum, high conductivity metal oxides (e.g., indium tin oxide, zincoxide, tungsten oxide) are capable of supporting surface plasmonresonance at their surfaces.

Optionally, a single nucleic acid molecule, or multiple clonal copies ofa nucleic acid, are attached to a nanoparticle, such that binding ofpolymerase to the nucleic acid causes a shift in the localized surfaceplasmon resonance (LSPR). Light incident on the nanoparticles inducesthe conduction electrons in them to oscillate collectively with aresonant frequency that depends on the nanoparticles' size, shape andcomposition. Nanoparticles of interest may assume different shapes,including spherical nanoparticles, nanorods, nanopyramids, nanodiamonds,and nanodiscs. As a result of these LSPR modes, the nanoparticles absorband scatter light so intensely that single nanoparticles are easilyobserved by eye using dark-field (optical scattering) microscopy. Forexample, a single 80-nm silver nanosphere scatters 445-nm blue lightwith a scattering cross-section of 3×10⁻² m², a million-fold greaterthan the fluorescence cross-section of a fluorescein molecule, and athousand fold greater than the cross-section of a similarly sizednanosphere filled with fluorescein to the self-quenching limit.Optionally, the nanoparticles are plasmon-resonant particles configuredas ultra-bright, nanosized optical scatters with a scattering peakanywhere in the visible spectrum. Plasmon-resonant particles areadvantageous as they do not bleach. Optionally, plasmon-resonantparticles are prepared, coated with template nucleic acids, and providedin a reaction mixture including a polymerase and one or morenucleotides, wherein a polymerase-template nucleic acid-particleinteraction is detected. One or more of the aforementioned steps may bebased on or derived from one or more methods disclosed by Schultz etal., in PNAS 97:996-1001 (2000), which is incorporated by referenceherein in its entirety.

The very large extinction coefficients at resonant wavelength enablesnoble-metal nanoparticles to serve as extremely intense labels fornear-surface interactions. Optionally, polymerase interaction withnanoparticle-localized DNA results in a shift in the resonantwavelength. The change in resonant wavelength due to binding or bindinginteractions can be measured in one of many ways. Optionally, theillumination is scanned through a range of wavelengths to identify thewavelength at which maximum scattering is observed at an imaging device.Optionally, broadband illumination is utilized in conjunction with adispersive element near the imaging device, such that the resonant peakis identified spectroscopically. Optionally, the nanoparticle system maybe illuminated at its resonant wavelength, or near its resonantwavelength, and any binding interactions may be observed as a drop inintensity of light scattered as the new resonant wavelength shifts awayfrom the illumination wavelength. Depending on the positioning of theilluminating wavelength, interactions may even appear as an increase innanoparticle scattering as the resonance peak shifts towards theillumination wavelength. Optionally, DNA-attached-nanoparticles may belocalized to a surface, or, alternatively, theDNA-attached-nanoparticles may be suspended in solution. A comprehensivereview of biosensing using nanoparticles is described by Anker et al.,in Nature Materials 7: 442-453 (2008), which is incorporated in itsentirety herein by reference.

Optionally, nano-features capable of LSPR are lithographically patternedon a planar substrate. The two dimensional patterning of nano-featureshas advantages in multiplexing and high-throughput analysis of a largenumber of different nucleic acid molecules. Optionally, gold nanopostsare substrates for surface plasmon resonance imaging detection ofpolymerase-template nucleic acid interactions, wherein the nucleic acidsare attached to the nanoposts. Nanostructure size and period caninfluence surface plasmon resonance signal enhancement, optionally,providing a 2, 3, 4, 5, 6, 7, 8-fold or higher signal amplification whencompared to control films.

Optionally, surface plasmon resonance may be sustained in planarsurfaces. A number of commercial instruments based on the Kretschmannconfiguration (e.g., Biacore, Uppsala, Sweden) and surface plasmonresonance imaging (e.g., GWC Technologies; Madison, WI; or Horiba;Kyoto, Japan) are available and have well established protocols forattaching DNA to their surfaces, as single spots and in multiplexedarray patterns. In the Kretschmann configuration, a metal film,typically gold, is evaporated onto the side of a prism and incidentradiation is launched at an angle to excite the surface plasmons. Anevanescent wave penetrates through the metal film exciting plasmons onthe other side, where it may be used to monitor near-surface and surfaceinteractions near the gold film. At the resonant angle, the lightreflected from the prism-gold interface is severely attenuated. Assumingfixed wavelength illumination, binding interactions may be examined bymonitoring both the intensity of the reflected light at a fixed angleclose to the resonant angle, as well as by monitoring the changes inangle of incidence required to establish surface plasmon resonanceconditions (minimum reflectivity). When a 2D imaging device such as aCCD or CMOS camera is utilized to monitor the reflected light, theentire illumination area may be imaged with high resolution. This methodis called surface plasmon resonance imaging (SPRi). It allows highthroughput analysis of independent regions on the surfacesimultaneously. Broadband illumination may also be used, in a fixedangle configuration, wherein the wavelength that is coupled to thesurface plasmon resonance is identified spectroscopically by looking fordips in the reflected spectrum. Surface interactions are monitoredthrough shifts in the resonant wavelength.

Surface plasmon resonance is a well-established method for monitoringprotein- nucleic acid interactions, and there exist many standardprotocols both for nucleic acid attachment as well as for analyzing thedata. Illustrative references from the literature include Cho et al.,“Binding Kinetics of DNA-Protein Interaction Using Surface PlasmonResonance,” Protocol Exchange, May 22, 2013; and Brockman et al., “AMultistep Chemical Modification Procedure To Create DNA Arrays on GoldSurfaces for the Study of Protein-DNA Interactions with Surface PlasmonResonance Imaging,” Journal of the American Chemical Society 121:8044-51 (1999), both of which are incorporated by reference herein intheir entireties.

Polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining localized surface plasmons. Optionally,polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining surface plasmon polaritons.

Optionally, polymerase/nucleic-acid interactions may be monitored onnanostructured surfaces capable of sustaining localized surfaceplasmons. Optionally, polymerase/nucleic-acid interactions may bemonitored on nanostructured surfaces capable of sustaining surfaceplasmon polaritons.

Optionally, extraordinary optical transmission (EOT) through a nanoholesarray may be used to monitor nucleic-acid/polymerase interactions. Lighttransmitted across subwavelength nanoholes in plasmonic metal films ishigher than expected from classical electromagnetic theory. Thisenhanced optical transmission may be explained by considering plasmonicresonant coupling to the incident radiation, whereby at resonantwavelength, a larger than anticipated fraction of light is transmittedacross the metallic nanoholes. The enhanced optical transmission isdependent on the dimensions and pitch of the nanoholes, properties ofthe metal, as well as the dielectric properties of the medium on eitherside of the metal film bearing the nanoholes. In the context of abiosensor, the transmissivity of the metallic nanohole array depends onthe refractive index of the medium contacting the metal film, whereby,for instance, the interaction of polymerase with nucleic acid attachedto the metal surface may be monitored as a change in intensity of lighttransmitted across the nanoholes array. Instrumentation and alignmentrequirements when using the EOT/plasmonic nanohole array approach ofsurface plasmon resonance may be employed using very compact optics andimaging elements. Low power LED illumination and a CMOS or CCD cameramay suffice to implement robust EOT plasmonic sensors. An exemplarynanohole array-based surface plasmon resonance sensing device isdescribed by Escobedo et al., in “Integrated Nanohole Array SurfacePlasmon Resonance Sensing Device Using a Dual-Wavelength Source,”Journal of Micromechanics and Microengineering 21: 115001 (2011), whichis herein incorporated by reference in its entirety.

The plasmonic nanohole array may be patterned on an optically opaquelayer of gold (greater than 50 nm thickness) deposited on a glasssurface. Optionally, the plasmonic nanohole array may be patterned on anoptically thick film of aluminum or silver deposited on glass.Optionally, the nanohole array is patterned on an optically thick metallayer deposited on low refractive index plastic. Patterning plasmonicnanohole arrays on low refractive index plastics enhances thesensitivity of the device to refractive index changes by better matchingthe refractive indices on the two sides of the metal layer. Optionally,refractive index sensitivity of the nanohole array is increased byincreasing the distance between holes. Optionally, nanohole arrays arefabricated by replication, for example, by embossing, casting,imprint-lithography, or template-stripping. Optionally, nanohole arraysare fabricated by self-assembly using colloids. Optionally, nanoholearrays are fabricated by projection direct patterning, such as laserinterference lithography.

A nano-bucket configuration may be preferable to a nanoholeconfiguration. In the nanohole configuration, the bottom of thenano-feature is glass or plastic or other appropriate dielectric,whereas in the nano-bucket configuration, the bottom of the nano-feature includes a plasmonic metal. The nano-bucket array advantageouslyis relatively simple to fabricate while maintaining the transmissionsensitivity to local refractive index.

Optionally, the nanohole array plasmonic sensing is combined withlens-free holographic imaging for large area imaging in an inexpensivemanner. Optionally, a plasmonic biosensing platform includes a plasmonicchip with nanohole arrays, a light-emitting diode source configured toilluminate the chip, and a CMOS imager chip to record diffractionpatterns of the nanoholes, which is modulated by molecular bindingevents on the surface. The binding events may be the formation of aclosed-complex between a polymerase and a template nucleic acid in thepresence of a nucleotide.

The methods to functionalize surfaces (for nucleic acid attachment) forsurface plasmon resonance sensing may be directly applied to EOTnanohole arrays as both sensing schemes employ similar metal surfaces towhich nucleic acids need to be attached.

Optionally, the refractive index changes associated withpolymerase/nucleic acid interaction may be monitored on nanostructuredsurfaces that do not support plasmons. Optionally, guided mode resonancemay be used to monitor the polymerase/nucleic-acid interaction.Guided-mode resonance or waveguide-mode resonance is a phenomenonwherein the guided modes of an optical waveguide can be excited andsimultaneously extracted by the introduction of a phase-matchingelement, such as a diffraction grating or prism. Such guided modes arealso called “leaky modes,” as they do not remain guided and have beenobserved in one and two-dimensional photonic crystal slabs. Guided moderesonance may be considered a coupling of a diffracted mode to awaveguide mode of two optical structured placed adjacent or on top ofeach other. For instance, for a diffraction grating placed on top of anoptical waveguide, one of the diffracted modes may couple exactly intothe guided mode of the optical waveguide, resulting in propagation ofthat mode along the waveguide. For off-resonance conditions, no light iscoupled into the waveguide, so the structure may appear completelytransparent (if dielectric waveguides are used). At resonance, theresonant wavelength is strongly coupled into the waveguide and may becouple out of the structure depending on downstream elements from thegrating-waveguide interface. In cases where the grating coupler isextended over the entire surface of the waveguide, the light cannot beguided, as any light coupled in is coupled out at the next gratingelement. Therefore, in a grating waveguide structure, resonance isobserved as a strong reflection peak, whereas the structure istransparent to off-resonance conditions. The resonance conditions aredependent on angle, grating properties, polarization and wavelength ofincident light. For cases where the guided mode propagation is notpresent, for instance due to a grating couple to the entire surface ofthe waveguide, the resonant mode may also be called leaky-moderesonance, in light of the strong optical confinement and evanescentpropagation of radiation in a transverse direction from the waveguidelayer. Change in dielectric properties near the grating, for instancedue to binding of biomolecules affects the coupling into the waveguide,thereby altering the resonant conditions. Optionally, where nucleic acidmolecules are attached to the surface of grating waveguide structures,the polymerase/nucleic-acid interaction may be monitored as a change inwavelength of the leaky mode resonance.

A diffraction element may be used directly on a transparent substratewithout an explicit need for a waveguide element. The change inresonance conditions due to interactions near the grating nanostructuremay be monitored as resonant wavelength shifts in the reflected ortransmitted radiation.

Reflected light from a nucleic acid attached guided mode resonant sensormay be used to monitor the polymerase/nucleic-acid interaction. Abroadband illumination source may be employed for illumination, and aspectroscopic examination of reflected light could reveal changes inlocal refractive index due to polymerase binding.

Optionally, a broadband illumination may be used and the transmittedlight may be examined to identify resonant shifts due to polymeraseinteraction. A linearly polarized narrow band illumination may be used,and the transmitted light may be filtered through a cross-polarizer;wherein the transmitted light is completely attenuated due to thecrossed polarizers excepting for the leaky mode response whosepolarization is modified. This implementation converts refractive indexmonitoring to a simple transmission assay that may be monitored oninexpensive imaging systems. Published material describe the assembly ofthe optical components. See, Nazirizadeh et al., “Low-Cost Label-FreeBiosensors Using Photonic Crystals Embedded between Crossed Polarizers,”Optics Express 18: 19120-19128 (2010), which is incorporated herein byreference in its entirety.

In addition to nanostructured surfaces, plain, unstructured surfaces mayalso be used advantageously for monitoring refractive index modulations.Optionally, interferometry may be employed to monitor the interaction ofpolymerase with nucleic acid bound to an un-structured, opticallytransparent substrate. Nucleic acid molecules may be attached to the topsurface of a glass slide by any means known in the art, and the systemilluminated from the bottom surface of the glass slide. There are tworeflection surfaces in this configuration, one reflection from thebottom surface of the glass slide, and the other from the top surfacewhich has nucleic acid molecules attached to it. The two reflected wavesmay interfere with each other causing constructive or destructiveinterference based on the path length differences, with the wavereflected from the top surface modulated by the changes in dielectricconstant due to the bound nucleic acid molecules (and subsequently bythe interaction of polymerase with the bound nucleic acid molecules).With the reflection from the bottom surface unchanged, any binding tothe nucleic acid molecules may be reflected in the phase differencebetween the beams reflected from the top and bottom surfaces, which inturn affects the interference pattern that is observed. Optionally,bio-layer interferometry is used to monitor the nucleic acid/polymeraseinteraction. Bio-layer interferometry may be performed on commercialdevices such as those sold by Pall Forte Bio corporation (Menlo Park,Calif.).

Optionally, the reflected light from the top surface is selectivelychosen by using focusing optics. The reflected light from the bottomsurface is disregarded because it is not in the focal plane. Focusingonly on the nucleic-acid-attached top surface, the light collected bythe focusing lens includes a planar wave, corresponding to the partiallyreflected incident radiation, and a scattered wave, corresponding to theradiations scattered in the collection direction by molecules in thefocal plane. These two components may be made to interfere if theincident radiation is coherent. This scattering based interferometricdetection is extremely sensitive and can be used to detect down tosingle protein molecules.

Optionally, a field-effect transistor (FET) is configured as a biosensorfor the detection of a closed-complex. A gate terminal of the FET ismodified by the addition of template nucleic acids. The binding of apolymerase including a charged tag results in changes in electrochemicalsignals. Binding of a polymerase with a next correct nucleotide to thetemplate nucleic acid provides different signals than polymerase bindingto a template nucleic acid in the presence of other incorrectnucleotides, where each incorrect nucleotide may also provide adifferent signal. Optionally, polymerase interactions with a templatenucleic acid are monitored using FET without the use of a an exogenouslabel on the polymerase, primed template nucleic acid, or nucleotide.Optionally, the pH change that occurs due to release of H⁺ions duringthe incorporation reaction is detected using a FET. Optionally, thepolymerase includes a tag that generates continuous H⁺ions that isdetected by the FET. Optionally, the continuous H⁺ion generating tag isan ATP synthase. Optionally, the continuous H⁺ion generation tag ispalladium, copper or another catalyst. Optionally, the release of a PPiafter nucleotide incorporation is detected using FET. For example, onetype of nucleotide may be provided to a reaction at a time. Once thenext correct nucleotide is added and conditions allow for incorporation,PPi is cleaved, released, and detected using FET, therefore identifyingthe next correct nucleotide and the next base. Optionally, templatenucleic acids are bound to walls of a nanotube. Optionally, a polymeraseis bound to a wall of a nanotube. FET is advantageous for use as asequencing sensor due to its small size and low weight, making itappropriate for use as a portable sequencing monitoring component.Details of FET detection of molecular interactions are described by Kimet al., in “An FET-Type Charge Sensor for Highly Sensitive Detection ofDNA Sequence,” Biosensors and Bioelectronics, Microsensors andMicrosystems 20: 69-74 (2004), doi:10.1016/j.bios.2004.01.025; and byStar et al., in “Electronic Detection of Specific Protein Binding UsingNanotube FET Devices,” Nano Letters 3: 459-63 (2003),doi:10.1021/n10340172, which are incorporated by reference herein intheir entireties.

By way of example, the polymerase includes a fluorescent tag. To monitorpolymerase-nucleic acid interaction with high signal-to-noise,evanescent illumination or confocal imaging may be employed. Theformation of a closed-complex on localized template nucleic acids may beobserved as an increased fluorescence compared to the background, forinstance, whereas in some instances it may be also be observed as adecreased fluorescence due to quenching or change in local polarenvironment. Optionally, a fraction of polymerase molecules may betagged with a fluorophore while another fraction may be tagged with aquencher in the same reaction mixture; wherein, the formation ofclosed-complex on a localized, clonal population of nucleic acid isrevealed as decrease in fluorescence compared to the background. Theclonal population of nucleic acids may be attached to a support surfacesuch as a planar substrate, microparticle, or nanoparticle. Optionally,a polymerase is tagged with a fluorophore, luminophore,chemiluminophore, chromophore, or bioluminophore.

Optionally, a plurality of template nucleic acids is tethered to asurface and one (or more) dNTPs are flowed in sequentially. The spectrumof affinities reveals the identity of the next correct nucleotide andtherefore the next base in the template nucleic acid. Optionally, theaffinities are measured without needing to remove and replace reactionmixture conditions (i.e., a wash step). Autocorrelation of the measuredintensities of the binding interaction, for instance, could readilyreveal the dynamics of nucleic acid sequence. Optionally, examinationincludes monitoring the affinity of the polymerase to the primedtemplate nucleic acid in the presence of nucleotides. Optionally, thepolymerase binds transiently with the nucleic acid and the bindingkinetics and affinity provides information about the identity of thenext base on the template nucleic acid. Optionally, a closed-complex isformed, wherein the reaction conditions involved in the formation of theclosed-complex provide information about the next base on the nucleicacid. Optionally, the polymerase is trapped at the polymerization sitein its the interaction, thus revealing the affinities without requiringa washing step to measure the off-rate.

Any technique that can measure dynamic interactions between a polymeraseand nucleic acid may be used to measure the affinities and enable thesequencing reaction methods disclosed herein.

Systems for Detecting Nucleotide-Specific Ternary Complex Formation

The provided methods can be performed using a platform, where anycomponent of the nucleic acid polymerization reaction is localized to asurface. Optionally, the template nucleic acid is attached to a planarsubstrate, a nanohole array, a microparticle, or a nanoparticle.Optionally, all reaction components are freely suspended in the reactionmixture, and not immobilized to a solid support substrate.

Optionally, the template nucleic acid is immobilized to a surface. Thesurface may be a planar substrate, a hydrogel, a nanohole array, amicroparticle, or a nanoparticle. Optionally, the reaction mixturescontain a plurality of clonally amplified template nucleic acidmolecules. Optionally, the reaction mixtures contain a plurality ofdistinguishable template nucleic acids.

Provided herein, inter alia, are systems for performing sequencingreactions involving the examination of the interaction between apolymerase and a primed template nucleic acid in the presence ofnucleotides to identify the next base in the template closed-complex byone or a combination of means including, but not limited to,crosslinking of the polymerase domains, crosslinking of the polymeraseto the nucleic acid, allosteric inhibition by small molecules,uncompetitive inhibitors, competitive inhibitors, non-competitiveinhibitors, and denaturation; wherein the formation of the trappedpolymerase complex provides information about the identity of the nextbase on the nucleic acid template.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanostructure. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanostructure. For example, the systemprovides a nanostructure, such as a chip, configured for use withsurface plasmon resonance to determine binding kinetics. An example ofsuch a chip is a CM5 Sensor S chip (GE Healthcare; Piscatawany, N.J.).The system may provide instrumentation such as a surface plasmonresonance instrument. The system may provide streptavidin and/or biotin.Optionally, the system provides biotin-DNA, DNA ligase, buffers, and/orDNA polymerase for preparation of biotinylated template DNA. Optionally,the system provides a gel or reagents (e.g., phenol:chloroform) forbiotinylated DNA purification. Alternatively, the system providesreagents for biotinylated template DNA characterization, for example,mass spectrometry or HPLC. Optionally, the system includes streptavidin,a chip, reagents, instrumentation, and/or instructions forimmobilization of streptavidin on a chip. Optionally, a chip is providedin the system already configured for template DNA coating, wherein thechip is immobilized with a reagent capable of binding template nucleicacids or modified template nucleic acids (e.g., biotinylated templateDNA). Optionally, the system provides reagents for chip regeneration.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanoparticle. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanoparticle. The nanoparticle may beconfigured for the electrochemical detection of nucleic acid-polymeraseinteraction, for instance, by using gold nanoparticles. Optionally, theDNA-nanoparticle conjugates are formed between aqueous gold colloidsolutions and template DNA molecules including, for example, free thiolor disulfide groups at their ends. The conjugates may include samenucleic acid sequence. Optionally, the nanoparticle conjugates arestabilized against flocculation and precipitation at high temperature(e.g., greater than 60° C.) and high ionic strength (e.g., 1M Na⁺).Optionally, the system provides reagents for preparing template DNAmolecules for nanoparticle attachment, including, generating templateDNA molecules with disulfides or thiols. Disulfide-containing templatenucleic acids may be synthesized using, for example, a 3′-thiol modifiercontrolled-pore glass (CPG) or by beginning with a universal support CPGand adding a disulfide modifier phosphoramidite as the first monomer inthe sequence. The system may provide nucleic acid synthesis reagentsand/or instructions for obtaining disulfide-modified template nucleicacids. Thiol-containing template nucleic acids may also be generatedduring nucleic acid synthesis with a 5′-tritylthiol modifierphosphoramidite. The system may provide reagents and/or instructions fornanoparticle conjugate purification using for example, electrophoresisor centrifugation. Optionally, nanoparticle conjugates are used tomonitor polymerase-template nucleic acid interactions colorimetrically.In this instance, the melting temperature of the nanoparticle conjugateincreases in the presence of strong polymerase binding. Therefore, thestrength of DNA binding can be determined by the change in this meltingtransition, which is observable by a color change. The systemsoptionally include reagents and equipment for detection of the meltingtransition.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, using adetectable polymerase. Optionally, the polymerase is detectably labeled.Optionally, the polymerase is detected using intrinsic properties of thepolymerase, for example, aromatic amino acids. Optionally, thepolymerase and template nucleic acids present in the system areconfigured for use in solution, without conjugation to a support. Thedetectable label on the polymerase may be a fluorophore, whereinfluorescence is used to monitor polymerase-template nucleic acid bindingevents. Optionally, the detectable polymerase may be used in combinationwith template nucleic acids in solution, or template nucleic acidsconjugated to a support structure. Optionally, one or more cysteineresidues of the polymerase is labeled with Cy3-maleimide. Optionally,the system includes reagents and/or instructions necessary to preparefluorescently labeled polymerase molecules. The system may includereagents and/or instructions for purification of fluorescently labeledpolymerases.

Procedural Features of the Methods

Following the examination step, where the identity of the next base hasbeen identified via formation of a closed-complex, the reactionconditions may be reset, recharged, or modified as appropriate, inpreparation for the optional incorporation step or an additionalexamination step. Optionally, the identity of the next base has beenidentified without chemically incorporating a nucleotide. Optionally,the identity of the next base is identified with chemical incorporationof a nucleotide, wherein a subsequent nucleotide incorporation has beeninhibited. Optionally, all components of the examination step, excludingthe template nucleic acid being sequenced, are removed or washed away,returning the system to the pre-examination condition. Optionally,partial components of the examination step are removed. Optionally,additional components are added to the examination step.

Optionally, reversible terminator nucleotides are used in theincorporation step to ensure one, and only one nucleotide isincorporated per cycle. No labels are required on the reversibleterminator nucleotides as the base identity is known from theexamination step. Non-fluorescently labeled reversible terminators arereadily available from commercial suppliers. Non-labeled reversibleterminator nucleotides are expected to have much faster incorporationkinetics compared to labeled reversible terminators due to their smallersteric footprint, and similar size to natural nucleotides.

Disclosed herein, in part, are reagent cycling sequencing methods,wherein sequencing reagents are introduced, one after another, for everycycle of examination and/or incorporation. Optionally, the sequencingreaction mixture includes a polymerase, a primed template nucleic acid,and at least one type of nucleotide. Optionally, the nucleotide and/orpolymerase are introduced cyclically to the sequencing reaction mixture.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. Optionally,a plurality of nucleotides and/or a plurality of polymerases areintroduced cyclically to the sequencing reaction mixture. Optionally,the examination step of the sequencing reaction has a differentcomposition than the incorporation step of the sequencing reaction.

Optionally, one or more nucleotides are sequentially added to andremoved from the sequencing reaction. Optionally, 1, 2, 3, 4, or moretypes of nucleotides are added to and removed from the reaction mixture.For example, one type of nucleotide is added to the sequencing reaction,removed, and replaced by another type of nucleotide. Optionally, anucleotide type present during the examination step is different from anucleotide type present during the incorporation step. Optionally, anucleotide type present during one examination step is different from anucleotide type present during a sequential examination step (i.e., thesequential examination step is performed prior to an incorporationstep). Optionally, 1, 2, 3, 4 or more types of nucleotides are presentin the examination reaction mixture and 1, 2, 3, 4, or more types ofnucleotides are present in the incorporation reaction mixture.

Optionally, a polymerase is cyclically added to and removed from thesequencing reaction. One or more different types of polymerases may becyclically added to and removed from the sequencing reaction.Optionally, a polymerase type present during the examination step isdifferent from a polymerase type present during the incorporation step.A polymerase type present during one examination step may be differentfrom a polymerase type present during a sequential examination step(i.e., the sequential examination step is performed prior to anincorporation step).

Optionally, conditions such as the presence of reagents, pH,temperature, and ionic strength are varied throughout the sequencingreaction. Optionally, a metal is cyclically added to and removed fromthe sequencing reaction. For example, a catalytic metal ion may beabsent during an examination step and present during an incorporationstep. Alternatively, a polymerase inhibitor may be present during anexamination step and absent during an incorporation step. Optionally,reaction components that are consumed during the sequencing reaction aresupplemented with the addition of new components at any point during thesequencing reaction.

Nucleotides can be added one type at a time, with the polymerase, to areaction condition that favors closed-complex formation. The polymerasebinds only to the template nucleic acid if the next correct nucleotideis present. A wash step after every nucleotide addition ensures allexcess polymerases and nucleotides not involved in a closed-complex areremoved from the reaction mixture. If the nucleotides are added one at atime, in a known order, the next base on the template nucleic acid isdetermined by the formation of a closed-complex when the addednucleotide is the next correct nucleotide. The closed-complex may beidentified by both the conformational change and the increased stabilityof the polymerase-template nucleic acid-nucleotide interaction.Optionally, the stability of the closed-complex formed in the presenceof the next correct nucleotide is at least an order of magnitude greaterthan the unstable interactions of the polymerase with the templatenucleic acid in the presence of incorrect nucleotides. The use of a washstep ensures that there are no unbound nucleotides and polymerases andthat the only nucleotides present in the reaction are those sequesteredin a closed-complex with a polymerase and a template nucleic acid. Oncethe next base on the template nucleic acid is determined, the nextcorrect nucleotide sequestered in the closed-complex may be incorporatedby flowing in reaction conditions that favor dissociation ordestabilization of the closed-complex and extending the template nucleicacid primer strand by one base (incorporation). Therefore, the wash stepensures that the only nucleotide incorporated is the next correctnucleotide from the closed-complex. This reagent cycling method may berepeated and the nucleic acid sequence determined. This reagent cyclingmethod may be applied to a single template nucleic acid molecule, or tocollections of clonal populations such as PCR products or rolling-circleamplified DNA. Many different templates can be sequenced in parallel ifthey are arrayed, for instance, on a solid support. Optionally, the washstep destabilizes binary complex formation.

Optionally, the washing is performed for a duration of time that ensuresthat the binary complex is removed, leaving the stabilizedclosed-complex in the reaction mixture. Optionally, the wash stepincludes washing the reaction with a high ionic strength or a high pHsolution.

Optionally, the incorporation step is a three stage process. In thefirst stage, all four nucleotide types are introduced into a reactionincluding a primed template nucleic acid, with a high fidelitypolymerase, in reaction conditions which favor the formation of aclosed-complex, and the next correct nucleotides are allowed to formstable closed-complexes with the template nucleic acid. In a secondstage, excess nucleotides and unbound polymerase are washed away. In athird stage, reaction conditions are modified so that the closed-complexis destabilized and the sequestered nucleotides within theclosed-complex become incorporated into the 3′-end of the templatenucleic acid primer. In an alternative approach, the second stage ismodified to remove completely any of the high fidelity polymerase andcognate nucleotide that may have been present in the closed-complex, andthe removed components are then replaced with a second polymerase andone or more nucleotides (e.g., reversible terminator nucleotides).Formation of tight polymerase-nucleic acid complexes in theincorporation step can be enabled by standard techniques such as fusinga non-specific DNA binding domain to the polymerase (e.g., the Phusionpolymerase, which is available from Thermo Fisher Scientific; Waltham,Mass.), and utilizing high concentrations of nucleotides to ensurecorrect nucleotides are always present in the closed-complex.

Polymerase molecules bind to primed template nucleic acid molecules in afingers-closed conformation in the presence of the next correctnucleotide even in the absence of divalent metal ions that are typicallyrequired for polymerase synthesis reactions. The conformational changetraps the nucleotide complementary to the next template base within theactive site of the polymerase. Optionally, the formation of theclosed-complex may be used to determine the identity of next base on thetemplate nucleic acid. Optionally, the primed template nucleic acids maybe contacted serially by different nucleotides in the presence ofpolymerase, in the absence of catalytic divalent metal ions; wherein theformation of a closed-complex indicates the nucleotide currently incontact with the template nucleic acid is the complementary nucleotideto the next base on the nucleic acid. A known order of nucleotides (inthe presence of polymerase and absence of catalytic metal ions) broughtinto contact with the template nucleic acid ensures facileidentification of the complementary nucleotide based on the particularposition in the order that induces closed-complex formation. Optionally,an appropriate wash step may be performed after every nucleotideaddition to ensure removal of all excess enzymes and nucleotides,leaving behind only the polymerase that is bound to nucleic acids in aclosed-complex with the next correct nucleotide at the active site. Theclosed-complex may be identified by means that reveal the conformationalchange of the polymerase in the closed conformation or by means thatreveal the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex compared tobinary polymerase-nucleic acid complexes or compared to unstableinteractions between the polymerase, primed template nucleic acid andincorrect nucleotides.

Optionally, the process of identifying the next complementary nucleotide(examination step) includes the steps of contacting immobilized primedtemplate nucleic acids with an examination mixture including polymeraseand nucleotides of one kind under conditions that inhibit the chemicalincorporation of the nucleotide, removing unbound reagents by a washstep, detecting the presence or absence of polymerase closed-complex onthe immobilized nucleic acids, and repeating these steps serially, withnucleotides of different kinds until a closed-complex formation isdetected. The closed-complex may be identified by both theconformational change and the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex. The wash stepbetween successive nucleotide additions may be eliminated by the use ofdetection mechanisms that can detect the formation of the closed-complexwith high fidelity, for instance, evanescent wave sensing methods ormethods that selectively monitor signals from the closed-complex. Theexamination steps noted above may be followed by an incorporation stepincluding, contacting the closed-complex with catalytic metal ions tocovalently add the nucleotide sequestered in the closed-complex to the3′-end of the primer. Optionally, the incorporation step may include,contacting the immobilized nucleic acids with a pre-incorporationmixture including a combination of multiple types of nucleotides andpolymerase under conditions that inhibit the chemical incorporation ofthe nucleotides; wherein the pre-incorporation mixture may containadditives and solution conditions to ensure highly efficientclosed-complex formation (e.g., low-salt conditions). The methods mayalso include performing a wash step to remove unbound reagents andproviding the immobilized complexes with an incorporation mixture,including catalytic metal ions, to chemically incorporate nucleotidessequestered within the active site of the polymerase. Thepre-incorporation mixture ensures highly efficient closed-complexformation, while the wash step and incorporation mixture ensure theaddition of a single nucleotide to the 3′-end of the primer. Optionally,the incorporation step may occur directly after examination an additionof one type of nucleotide. For instance, a repeated pattern used forsequencing may include the following flow pattern (i) dATP+/polymerase,(ii) Wash, (iii) Mg⁺², (iv) Wash, (v) dTTP+/polymerase, (vi) Wash, (vii)Mg⁺², (viii) Wash, (ix) dCTP+/polymerase, (x) Wash (xi) Mg⁺², (xii)Wash, (xiii) dGTP+/polymerase, (xiv) Wash, (xv) Mg⁺², (xvi)Wash.Optionally, the repeated pattern used for sequencing may include (i)dATP+/polymerase, (ii) Wash, (iii) dTTP+/polymerase, (iv) Wash, (v)dGTP+/polymerase, (vi) Wash, (vii) dCTP+/polymerase, (viii) Wash, (ix)Pre-incorporation mixture, (x) Wash, (xi) Mg, (xii)Wash. The wash stepstypically contain metal ion chelators and other small molecules toprevent accidental incorporations during the examination steps. Afterthe incorporation step, the primer strand is typically extended by onebase. Repeating this process, sequential nucleobases of a nucleic acidmay be identified, effectively determining the nucleic acid sequence.Optionally, the examination step is performed at high salt conditions,for example, under conditions of 50 mM to 1,500 mM salt.

For sequencing applications, it can be advantageous to minimize oreliminate fluidics and reagents exchange. Removing pumps, valves andreagent containers can allow for simplified manufacturing of smallerdevices. Disclosed herein, in part, are “all-in” sequencing methods,wherein the need to introduce reagents one after another, for everycycle of examination and/or incorporation, is eliminated. Reagents areadded only once to the reaction, and sequencing-by-synthesis isperformed by manipulating reagents already enclosed within thesequencing reaction. A scheme such as this requires a method todistinguish different nucleotides, a method to synchronize incorporationof nucleotides across a clonal population of nucleic acids and/or acrossdifferent nucleic acid molecules, and a method to ensure only onenucleotide is added per cycle.

Optionally, the sequencing reaction mixture includes a polymerase, aprimed template nucleic acid, and at least one type of nucleotide.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. As providedherein, a polymerase refers to a single polymerase or a plurality ofpolymerases. As provided herein, a primed template nucleic acid ortemplate nucleic acid refers to a single primed template nucleic acid orsingle template nucleic acid, or a plurality of primed template nucleicacids or a plurality of template nucleic acids. As provided herein, anucleotide refers to one nucleotide or a plurality of nucleotides. Asprovided herein, a single nucleotide is one nucleotide. Optionally, thesequencing reaction nucleotides include, but are not limited to, 1, 2,3, or 4 of the following nucleotides: dATP, dGTP, dCTP, dTTP, and dUTP.

Optionally, the examination step and the incorporation step take placein a single sequencing reaction mixture.

Optionally, 1, 2, 3, 4 or more types of nucleotides (e.g., dATP, dGTP,dCTP, dTTP) are present in the reaction mixture together at the sametime, wherein one type of nucleotide is a next correct nucleotide. Thereaction mixture further includes at least one polymerase and at leastone primed template nucleic acids. Optionally, the template nucleic acidis a clonal population of template nucleic acids. Optionally, thepolymerase, primed template nucleic acid, and the nucleotide form aclosed-complex under examination reaction conditions.

In the provided methods, four types of nucleotides can be present atdistinct and different concentrations wherein the diffusion and bindingtimes of the polymerase to the template nucleic acid are different foreach of the four nucleotides, should they be the next correctnucleotide, due to the different concentrations of the four nucleotides.For example, the nucleotide at the highest concentration would bind toits complementary base on the template nucleic acid at a fast time, andthe nucleotide at the lowest concentration would bind to itscomplementary base on the template nucleic acid at a slower time;wherein binding to the complementary base on the template nucleic acidrefers to the polymerase binding to the template nucleic acid with thenext correct nucleotide in a closed closed-complex. The identity of thenext correct nucleotide is therefore determined by monitoring the rateor time of binding of polymerase to the template nucleic acid in aclosed-complex. Optionally, the four types of nucleotides may bedistinguished by their concentration, wherein the differentconcentrations of the nucleotides result in measurably differenton-rates for the polymerase binding to the nucleic acid. Optionally, thefour types of nucleotides may be distinguished by their concentration,wherein the different concentrations of the nucleotides result inmeasurably different on-rates for the formation of a stabilizedclosed-complex.

Optionally, the polymerase is labeled. In some instances, the polymeraseis not labeled (i.e., does not harbor an exogenous label, such as afluorescent label) and any label-free detection method disclosed hereinor known in the art is employed. Optionally, the binding of thepolymerase to the nucleic acid is monitored via a detectable feature ofthe polymerase. Optionally, the formation of a stabilized closed-complexis monitored via a detectable feature of the polymerase. A detectablefeature of the polymerase may include, but is not limited to, optical,electrical, thermal, colorimetric, mass, and any combination thereof.

Optionally, 1, 2, 3, 4, or more nucleotides types (e.g., dATP, dTTP,dCTP, dGTP) are tethered to 1, 2, 3, 4, or more different polymerases;wherein each nucleotide type is tethered to a different polymerase andeach polymerase has a different exogenous label or a detectable featurefrom the other polymerases to enable its identification. All tetherednucleotide types can be added together to a sequencing reaction mixtureforming a closed-complex including a tethered nucleotide-polymerase; theclosed-complex is monitored to identify the polymerase, therebyidentifying the next correct nucleotide to which the polymerase istethered. The tethering may occur at the gamma phosphate of thenucleotide through a multi-phosphate group and a linker molecule. Suchgamma-phosphate linking methods are standard in the art, where afluorophore is attached to the gamma phosphate linker. Optionally,different nucleotide types are identified by distinguishable exogenouslabels. Optionally, the distinguishable exogenous labels are attached tothe gamma phosphate position of each nucleotide.

Optionally, the sequencing reaction mixture includes a catalytic metalion. Optionally, the catalytic metal ion is available to react with apolymerase at any point in the sequencing reaction in a transientmanner. To ensure robust sequencing, the catalytic metal ion isavailable for a brief period of time, allowing for a single nucleotidecomplementary to the next base in the template nucleic acid to beincorporated into the 3′-end of the primer during an incorporation step.In this instance, no other nucleotides, for example, the nucleotidescomplementary to the bases downstream of the next base in the templatenucleic acid, are incorporated. Optionally, the catalytic metal ionmagnesium is present as a photocaged complex (e.g., DM-Nitrophen) in thesequencing reaction mixture such that localized UV illumination releasesthe magnesium, making it available to the polymerase for nucleotideincorporation. Furthermore, the sequencing reaction mixture may containEDTA, wherein the magnesium is released from the polymerase active siteafter catalytic nucleotide incorporation and captured by the EDTA in thesequencing reaction mixture, thereby rendering magnesium incapable ofcatalyzing a subsequent nucleotide incorporation.

Thus, in the provided methods, a catalytic metal ion can be present in asequencing reaction in a chelated or caged form from which it can bereleased by a trigger. For example, the catalytic metal ion catalyzesthe incorporation of the closed-complex next correct nucleotide, and, asthe catalytic metal ion is released from the active site, it issequestered by a second chelating or caging agent, disabling the metalion from catalyzing a subsequent incorporation. The localized release ofthe catalytic metal ion from its cheating or caged complex is ensured byusing a localized uncaging or un-chelating scheme, such as an evanescentwave illumination or a structured illumination. Controlled release ofthe catalytic metal ions may occur for example, by thermal means.Controlled release of the catalytic metal ions from their photocagedcomplex may be released locally near the template nucleic acid byconfined optical fields, for instance by evanescent illumination such aswaveguides or total internal reflection microscopy. Controlled releaseof the catalytic metal ions may occur for example, by altering the pH ofthe solution near the vicinity of the template nucleic acid. Chelatingagents such as EDTA and EGTA are pH dependent. At a pH below 5, divalentcations Mg²⁺ and Mn²⁺ are not effectively chelated by EDTA. A method tocontrollably manipulate the pH near the template nucleic acid allows thecontrolled release of a catalytic metal ion from a chelating agent.Optionally, the local pH change is induced by applying a voltage to thesurface to which the nucleic acid is attached. The pH method offers anadvantage in that that metal goes back to its chelated form when the pHis reverted back to the chelating range.

Optionally, a catalytic metal ion is strongly bound to the active siteof the polymerase, making it necessary to remove the polymerase from thetemplate nucleic acid after a single nucleotide incorporation. Theremoval of polymerase may be accomplished by the use of a highlydistributive polymerase, which falls off the 3′-end of the strand beingsynthesized (e.g., primer) after the addition of every nucleotide. Theunbound polymerase may further be subjected to an electric or magneticfield to remove it from the vicinity of the nucleic acid molecules. Anymetal ions bound to the polymerase may be sequestered by chelatingagents present in the sequencing reaction mixture, or by molecules whichcompete with the metal ions for binding to the active site of thepolymerase without disturbing the formation of the closed-complex. Theforces which remove or move the polymerase away from the templatenucleic acid (e.g., electric field, magnetic field, and/or chelatingagent) may be terminated, allowing for the polymerase to approach thetemplate nucleic acid for another round of sequencing (i.e., examinationand incorporation). The next round of sequencing, in a non-limitingexample, includes the formation of a closed-complex, removing unboundpolymerase away from the vicinity of the template nucleic acid and/orclosed-complex, controlling the release of a catalytic metal ion toincorporate a single nucleotide sequestered within the closed-complex,removing the polymerase which dissociates from the template nucleic acidafter single incorporation away from the vicinity of the templatenucleic acid, sequestering any free catalytic metal ions through the useof chelating agents or competitive binders, and allowing the polymeraseto approach the template nucleic acid to perform the next cycle ofsequencing.

Described above are polymerase-nucleic acid binding reactions for theidentification of a nucleic acid sequence. However, nucleic acidsequence identification may include information regarding nucleic acidmodifications, including methylation and hydroxymethylation. Methylationmay occur on cytosine bases of a template nucleic acid. DNA methylationmay stably alter the expression of genes. DNA methylation is alsoindicated in the development of various types of cancers,atherosclerosis, and aging. DNA methylation therefore can serve as anepigenetic biomarker for human disease.

Optionally, one or more cytosine methylations on a template nucleic acidare identified during the sequencing-by-binding methods provided herein.The template nucleic acid may be clonally amplified prior to sequencing,wherein the amplicons include the same methylation as their templatenucleic acid. Amplification of the template nucleic acids may includethe use of DNA methyltransferases to achieve amplicon methylation. Thetemplate nucleic acids or amplified template nucleic acids are providedto a reaction mixture including a polymerase and one or more nucleotidetypes, wherein the interaction between the polymerase and nucleic acidsis monitored. Optionally, the interaction between the polymerase andtemplate nucleic acid in the presence of a methylated cytosine isdifferent than the interaction in the presence of an unmodifiedcytosine. Therefore, based on examination of a polymerase-nucleic acidinteraction, the identity of a modified nucleotide is determined.

Optionally, following one or more examination and/or incorporationsteps, a subset of nucleotides is added to reduce or reset phasing.Thus, the methods can include one or more steps of contacting a templatenucleic acid molecule being sequenced with a composition comprising asubset of nucleotides and an enzyme for incorporating the nucleotidesinto the strand opposite the template strand of the nucleic acidmolecule. The contacting can occur under conditions to reduce phasing inthe nucleic acid molecule. Optionally, the step of contacting thetemplate nucleic acid molecule occurs after an incorporation step and/orafter an examination step. Optionally, the contacting occurs after 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75,80, 85, 90, 95, or 100 rounds or more of sequencing, i.e., rounds ofexamination and incorporation. Optionally, the contacting occurs after30 to 60 rounds of sequencing. Optionally, the contacting occurs afterevery round of sequencing (i.e., after one set of examination andincorporation steps). Optionally, multiple contacting steps occur afterevery round of sequencing, wherein each contacting step may comprisedifferent subsets of nucleotides. Optionally, the method furthercomprises one or more washing steps after contacting. Optionally, thesubset comprises two or three nucleotides. Optionally, the subsetcomprises three nucleotides. Optionally, the subset of nucleotides isselected from three of dATP, dGTP, dCTP, dTTP or a derivative thereof.Optionally, the three nucleotides comprise adenosine, cytosine, andguanine. Optionally, the three nucleotides comprise adenosine, cytosine,and thymine. Optionally, the three nucleotides comprise cytosine,guanine and thymine. Optionally, the three nucleotides compriseadenosine, guanine and thymine. Optionally, each round of contactingcomprises the same subset or different subsets of nucleotides.Optionally, sequencing of a nucleic acid template is monitored and thecontacting with the subset of nucleotides occurs upon detection ofphasing. See also for example, U.S. Pat. No. 8,236,532, which isincorporated herein by reference in its entirety.

Optionally, the sequencing reaction involves a plurality of templatenucleic acids, polymerases and/or nucleotides, wherein a plurality ofclosed-complexes is monitored. Clonally amplified template nucleic acidsmay be sequenced together wherein the clones are localized in closeproximity to allow for enhanced monitoring during sequencing.Optionally, the formation of a closed-complex ensures the synchronicityof base extension across a plurality of clonally amplified templatenucleic acids. The synchronicity of base extension allows for theaddition of only one base per sequencing cycle.

EXAMPLES

Preliminary testing demonstrated how specific interaction of a primedtemplate nucleic acid, a polymerase, and the next correct nucleotide(i.e., the cognate nucleotide) formed a ternary complex that indicateduseful sequence information. Identity of the next correct nucleotide wasadvantageously determined before chemical incorporation into a primerextension product, and by the use of native nucleotides (e.g., unlabelednucleotides consisting of dATP, dGTP, dCTP, and dTTP). While modifiednucleotides (e.g., nucleotide analogs, or nucleotides incorporatingfluorescent or other labels) can be used in the procedure, nativenucleotides facilitated rapid cycling by eliminating the need to removelabels or other chemical moieties during the sequencing protocol. Asdiscussed above in connection with approaches for enhancingmatch/mismatch base discrimination, salt concentrations can bemanipulated to favor ternary complex formation during the examinationstep. This increased the signal-to-noise ratio resulting from ternarycomplex formation, and improved correct base calling. Other approachesfor improving correct base calling and extending read lengths aredescribed below.

FIG. 1 presents results obtained using a standard SBB procedure withhomopolymer compression that did not employ the biphasic protocol forbinding primed template nucleic acid with polymerase and nucleotide.Approximately 28 cycles of examination and incorporation of all fourdNTPs (using single base interrogations) were used to obtain just over100 nucleotides of sequence data. Measured signals (vertical axis)indicating ternary complex formation (associated with identification ofcognate nucleotides) exhibited a declining trend as the cycle number(horizontal axis) increased. Conversely, measured signals indicatingdetection of binary complexes (associated with identification ofnon-cognate nucleotides) did not similarly decline. More particularly,signal associated with background detection of binary complexes remainedsubstantially constant over the duration of the sequencing procedure. Asa result, the signal:noise ratios for correct base calling showed atrend that disfavored correct base calling from sequence reads longerthan illustrated in the figure. Accordingly, it was of interest toinvestigate methods that could be used to reduce background signalassociated with binary complex formation for the purpose of improvingsignal:noise ratios useful for base calling in the SBB procedure.

The following Examples illustrate a technique for comparing monitoredinteractions under two different polymerase binding conditions, wherethe polymerase concentration is held constant and a test nucleotide(e.g., dATP, dGTP, dCTP, dTTP; nucleotide analog; or labeled nucleotide)is present at two different concentrations. The two nucleotideconcentrations can be relatively lower and higher compared to eachother. For example, the two nucleotide concentrations can differ by 10fold, or more. In fact, the two different binding conditions can differby the presence and absence (i.e., zero concentration) of the testnucleotide(s). The nucleotide concentration used in the second of thetwo conditions will always be higher, so that the primed templatenucleic acid is exposed first to the polymerase and the lower of the twonucleotide concentrations. The term, “biphasic” is used below whenreferring to binding of a primed template nucleic acid with polymeraseand a nucleotide, where the nucleotide is first present at a lowerconcentration (or even zero concentration) and then at a higherconcentration. Results from the procedure unexpectedly indicated thatinteractions between DNA polymerase and the primed template nucleic acidwere very different in the binary and ternary complexes. As well, thetechnique was unexpectedly found to be useful for detecting homopolymerstretches in templates being sequenced.

Example 1 illustrates the advantages of contacting a primed templatenucleic acid with polymerase according to a biphasic protocol whenperforming sequencing-by-binding procedures. Rather than suppressingformation of binary complexes that might confound correct base calling,the approach relied on comparing binding signals when the concentrationof polymerase was constant and the nucleotide concentration wasincreased. The quality of the signal employed for base calling increaseddramatically by use of the biphasic protocol. More particularly, theprotocol essentially established a baseline of nonspecific polymerasebinding.

Example 1 Background Reduction Enhanced Detection of Nucleotide-SpecificInteraction Between DNA Polymerase and a Primed Template Nucleic Acid

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thebackground reduction technique. Template strands biotinylated at their5′-ends were used to immobilize the primed template nucleic acid ontofiber optic tips functionalized with streptavidin (SA) according tostandard procedures. The expected sequence read from the biotinylatedALK100 template DNA had the sequence of SEQ ID NO:1 (potential readlength of 86 nucleotides), and the hybridized DNA primer had thesequence of SEQ ID NO:2. Tips were washed in a Tris-buffered solutioncontaining 200 mM KCl, 160 mM potassium glutamate, and 0.01% Tween-20before commencing the cycling protocol. Independent binding reactionsfor each of four test nucleotides were carried out in serial fashionusing examination buffers that contained Tris-HCl (pH 8.0), 160 mM KCl,160 mM potassium glutamate, 0.01% Tween-20, 3% DMSO, 300 mM betaine, 1mM β-mercaptoethanol, 100 μg/m1 BSA, 1 mM NiSO₄, 280 U/ml Bsu DNApolymerase large fragment and one of the nucleotides at a concentrationof 100 μM (dCTP, dATP, and dGTP) or 400 μM (dTTP). Examination buffersin this procedure were supplemented with DMSO and betaine to control DNAsecondary structure. No wash steps were required between reagentchanges. When using a single contacting step to effect binding ofpolymerase and nucleotide to the primed template nucleic acid, thebinding step was 200 seconds long, with monitoring of bindinginteractions being continuous. This was accomplished by contacting theprimed template nucleic acid with a single solution that included thepolymerase and test nucleotide. In one test of the biphasic bindingprotocol, the procedure involved first contacting the primed templatenucleic acid with a solution that included the polymerase and onenucleotide at a 10 μM concentration for 45 seconds, and then contactingthe primed template nucleic acid with a solution that included thepolymerase and the same nucleotide at a higher concentration (100 μM fordCTP, dATP, dGTP; and 400 μM for dTTP) for 30 seconds. In another testof the biphasic binding protocol, the solution used in the firstcontacting step included the polymerase, but did not include any of thenucleotides being investigated for possible binding interactions; andthe solution used in the second contacting step included the polymeraseand one nucleotide (100 μM for dCTP, dATP, dGTP; and 400 μM for dTTP).In this latter test of the biphasic binding protocol, the first andsecond contacting steps were carried out for 15 seconds each. In bothtests of the biphasic binding protocol, the polymerase-containingsolutions used in the first and second contacting steps included thepolymerase at a constant concentration (i.e., 280 U/ml). Next there wasan incorporation step, whereby the next correct nucleotide included in aternary complex was chemically incorporated into the primer strand. Thiswas effected by transferring tips to a Tris-buffered solution thatincluded 50 mM KCl, 0.01% Tween-20, 3% DMSO, 300 mM betaine, 1 mMβ-mercaptoethanol, 100 μg/ml bovine serum albumin (BSA), 1 U/ml of theBsu DNA polymerase large fragment, and MgCl₂ at a final concentration of2 mM to promote the chemical incorporation. Next, there was a step forquenching the incorporation reaction by chelating the divalent cation.Quenching was effected by transferring tips from the incorporationbuffer to a Tris-buffered wash solution that included 500 mM KCl, 0.02%Tween-20, and 2 mM EDTA. Finally, each cycle was completed by soakingtips in a Tris-buffered regeneration solution that included 200 mM KCl,160 mM potassium glutamate, and 0.01% Tween-20. Results frominterferometry monitoring were analyzed to identify correct andincorrect nucleotides. In the case of results obtained from use of thebiphasic binding procedure, this involved comparing signals measuredafter the first and second contacting steps that involved polymerase,and required that the signal measured after the second contacting stepexceed the signal measured after the first contacting step to identifythe test nucleotide as the next correct nucleotide. For example, thesignal measured after the second contacting step must have exceeded thesignal measured after the first contacting step either by a minimumthreshold to be scored as the next correct nucleotide. Steps wererepeated to obtain extensive sequence information for the templatenucleic acid.

FIGS. 2A and 2B show, respectively, interferometry traces obtained usinga standard single binding step that involved contacting primed templatenucleic acid with polymerase and nucleotide simultaneously; and abiphasic binding step that involved contacting primed template nucleicacid first with polymerase and nucleotide at 10 μM, and then withpolymerase and nucleotide at the higher nucleotide concentration. Tracesfor cycle numbers 3 and 4 in FIG. 2A are very similar, even though cycle3 (conducted using dTTP) should be a negative result, while cycle 4 is apositive result (conducted using dGTP). This illustrates a difficultcall at cycle 3, and could generate a false-positive result indicating Tat the position monitored in cycle 3. Also of interest is that cycle 9was very similar to other true-positive calls, even though cycle 9actually represents detection of a homopolymer triplet (i.e., GGG). Incontrast the trace shown in FIG. 2B clearly distinguished positive callsfrom negative calls. In all instances, positive calls were indicated byhigher signals when the second (i.e., higher) concentration ofnucleotide was used. Conversely, negative calls did not show substantialchange between the maximum signal measured during the first contactingstep, and the signal measured during the second contacting step thatemployed the higher nucleotide concentration. Simply stated, a positiveresult was indicated when the monitored signal was higher in the secondcontacting step compared to the maximum signal monitored during thefirst contacting step. This also can be processed by requiring a minimumratio of signals measured in the second and first contacting steps.Alternatively, the slope of a line or vector connecting the first andlast points of a particular nucleotide exposure period can be analyzedto determine whether a minimum value was exceeded to make a positivebase call. Continuing with the data analysis, a doublet G starting atbase position 8 (being detected at cycle numbers 15-16) yielded anunusually high signal during the second contacting step, therebyindicating the biphasic binding procedure could distinguish unique,single bases from the presence of homopolymer stretches. Finally, thetraces in FIGS. 2A and 2B differed qualitatively with respect to theresponse observed during the incorporation step (i.e., second step foreach cycle of a single base in FIG. 2A; third step for each cycle of asingle base in FIG. 2B). More particularly, the signal uniformlyincreased for the duration of the incorporation step in FIG. 2A, butdecreased during the incorporation step in FIG. 2B. This differenceindicated that complexes formed by the different binding step approacheswere not the same. Stated differently, the biphasic binding approach wasnot a variant of the single-step binding approach that yielded the sameresult. Instead, profiles of the different trace patterns reflected amechanistic difference in the interaction between polymerase and theprimed template nucleic acid.

Example 2 describes techniques used to assess the quality of sequenceinformation obtained in a sequencing-by-binding procedure thatincorporated a biphasic polymerase binding protocol.

Example 2 Enhanced Detection of Ternary Complex Formation ImprovedAccuracy of Data Obtained in a Sequencing-by-Binding Protocol

Primed template nucleic acid was immobilized to fiber optic tips, andthe tips washed in preparation for use with the FORTEBIO® (Menlo Park,Calif.) Octet instrument essentially as described under Example 1.Compositions of the examination buffer, the incorporation buffer, thequenching buffer, and the regeneration buffer used in the procedure alsowere the same as in Example 1. A control trial conducted using a singlestep for contacting the primed template nucleic acid with polymerase wascarried out using the Bsu DNA polymerase large fragment in combinationwith one of the test nucleotides at concentrations of 100 μM (dCTP,dATP, and dGTP) or 400 μM (dTTP). The step of contacting polymerase andnucleotide to the primed template nucleic acid in this control trialproceeded for 200 seconds, with monitoring of binding interactions beingcontinuous. The trial incorporating biphasic polymerase binding to theprimed template nucleic acid employed a first contacting step using asolution that included the polymerase and one nucleotide at a 2μMconcentration for 45 seconds. This was followed by a second contactingstep using a solution that included the polymerase and the samenucleotide at a higher concentration (100 μM for dCTP, dATP, dGTP; and400 μM for dTTP) for 30 seconds. Solutions employed in the first andsecond contacting steps included the polymerase at a constantconcentration (i.e., 280 U/ml). Next there was an incorporation step,whereby the next correct nucleotide included in a ternary complex waschemically incorporated into the primer strand. This was effected bytransferring tips to a Tris-buffered solution that included 50 mM KCl,0.01% Tween-20, 3% DMSO, betaine, beta-mercaptoethanol, bovine serumalbumin (BSA), the Bsu DNA polymerase large fragment, and MgCl₂ at afinal concentration of 2 mM to promote the chemical incorporation.Notably, buffers used in the examination and incorporation steps weresupplemented with DMSO and betaine to control DNA secondary structure.Next, there was a step for chelating the divalent cation to quench theincorporation reaction. Quenching was effected by transferring tips fromthe incorporation buffer to a Tris-buffered wash solution that included500 mM KCl, 0.02% Tween-20, and 2 mM EDTA. Finally, each cycle wascompleted by soaking tips in a Tris-buffered regeneration solution thatincluded KCl, potassium glutamate, and Tween-20. Procedures foridentifying the next correct nucleotide based on monitoredinterferometry results were as described under Example 1. Steps forcontacting the primed template nucleic acid with the different solutionswere repeated to obtain sequence information for the template nucleicacid.

FIGS. 3A and 3B show, respectively, base calling results over a lengthof about 60 nucleotides for the same primed template nucleic acidsubjected to sequencing-by-binding procedures with homopolymercompression, wherein polymerase either in a single step, or using thebiphasic binding protocol. The plot in FIG. 3A includes onefalse-negative call, and one false-positive call in the sequence beyond50 nucleotides. Correct negative calls generally were associated withdetectable signals that were below an arbitrary threshold needed to calla positive result. Interestingly, the trend indicates a decrease insignal magnitude with increasing read length for correct positive calls,while the background signal associated with correct negative callsremained substantially stable. Consequently, the signal-to-noise ratiodecreased with increasing read length. If not reduced or controlled bysome means, there would be an upper limit to the read length. Incontrast, FIG. 3B shows that detectable signal associated with correctnegative calls were substantially eliminated by the biphasic polymerasebinding procedure. The signal-to-noise ratio for correct calls wasdramatically more favorable with the biphasic binding procedure. Stillfurther, no sequencing errors were detected in the results obtainedusing the biphasic binding procedure.

Using a biphasic binding technique that differed slightly from theprocedure described immediately above, correct base calling results wereachieved over 200 bases of read length with no errors. Morespecifically, primed template nucleic acid was first contacted with DNApolymerase in the absence of the test nucleotide, and then contactedwith the combination of DNA polymerase and the test nucleotide. Asabove, cycles were carried out for binding (i.e., first and secondcontacting steps employing polymerase-containing solutions),incorporation (i.e., addition of catalytic divalent cation), quenching(i.e., removing divalent cation and products of the polymerasereaction), and buffer regeneration. Results indicated that only twoerrors were observed over a read length of 230 nucleotides. Thisdramatic improvement illustrated the advantage of the biphasic bindingtechnique.

Example 3 demonstrates how the biphasic examination procedure was usedfor examining a plurality of test nucleotides before conducting anincorporation reaction. This technique advantageously reduced backgroundsignal associated with detection of binary complexes, thereby enhancingspecific detection of ternary complexes that included the next correctnucleotide. As well, assay time was shortened by avoiding unproductiveincorporation reactions. Nucleotides used in the incorporation reactioncan be native nucleotides or nucleotide analogs that are unlabeled.Alternatively, nucleotides in the incorporation reaction can benucleotide analogs that are labeled or unlabeled, and/or that include areversible terminator moiety.

Example 3 Progressive Binding and Examination of a Plurality of TestNucleotides Followed by Incorporation Reaction

Primed template nucleic acid was immobilized to fiber optic tips, andthe tips washed in preparation for use with the FORTEBIO® (Menlo Park,Calif.) Octet instrument essentially as described under Example 1, withimmobilization of the primed template nucleic acid proceeding over thecourse of 200 seconds. The sensor tip was then washed for 45 seconds inbinding buffer supplemented with 100 μg/ml of ultrapure bovine serumalbumin (BSA). Next, the tip was exposed to Examination Buffer (asolution containing Tris-HCl (pH 8.0), 160 mM KCl, 160 mM potassiumglutamate, 0.01% Tween-20, 100 μg/mL ultrapure BSA, 1 mM NiSO₄, and 400nM Bsu polymerase large fragment) for 30 seconds to allow for binarycomplex formation in the absence of dNTP. The tip was then exposed toExamination Buffer supplemented with a single dNTP (100 μM dTTP) for 20seconds; followed by a 20 second exposure to Examination Buffersupplemented with two dNTPs (100 μM dTTP and 100 μM dATP); followed by a20 second exposure to Examination Buffer supplemented with three dNTPs(100 μM dTTP, 100 μM dATP, and 100 μM dCTP); and finally followed by a20 second exposure to Examination Buffer supplemented with all fourdNTPs (100 μM for each of dTTP, dATP, dCTP and dGTP). The tip wasremoved from the final solution and plunged directly into an EDTA washbuffer (30 mM Tris-HCl pH 8.0, 500 mM KCl, 0.02% Tween-20, 2 mM EDTA)for 20 seconds to strip polymerase and nucleotides from any complexesthat had formed with the primed template nucleic acid. Following removalof polymerase, dNTPs, Ni²⁺, and potassium glutamate during the EDTAwash, the tip was equilibrated in an enzyme Incorporation Buffer thatdid not include either polymerase or nucleotides for a period of 20seconds. This equilibration step, which is optional, served to removefrom the tip any reagents that may have carried over from the precedingstep. The tip was then exposed for 5 seconds to Incorporation Buffersupplemented with a polymerase and one or more dNTP (e.g., a single dNTPif the next correct nucleotide is known, or all four reversibleterminator dNTPs), and an amount of a catalytic metal ion (e.g., Mg²⁺)sufficient to promote dNTP incorporation and extension of the primer inresponse to the next correct nucleotide of the template strand. The tipwas exposed again to the EDTA wash buffer for 20 seconds to removepolymerase and reversible terminators, and to an ExaminationRegeneration solution for 20 seconds prior to repeating the cyclestarting with the no dNTP examination step (i.e., the first phase of thebiphasic polymerase binding protocol). The Examination Regenerationsolution was a Tris-buffered solution that included 200 mM KCl, 160 mMpotassium glutamate, and 0.01% Tween-20. Monitoring was continuous inthis demonstration, although periodic monitoring also would have yieldedgood results. All steps were performed at 30° C., with shaking at 500rpm.

FIG. 4 shows an interferometry trace that clearly confirmed thedisclosed approach increased the signal:noise ratio for ternary complexdetection. The initial phase of polymerase binding, where the primedtemplate nucleic acid was contacted with the polymerase in the absenceof added nucleotide, led to a substantial increase in binding signalduring the 30 second treatment. This indicated formation of the binarycomplex between the primed template nucleic acid and the polymerase.Examination carried out in the presence of polymerase and dTTPsubstantially remained unchanged when compared with signal measured inthe preceding step. This indicated only binary complexes were formed inthe presence of dTTP, and that dTTP was not the next correct nucleotide.Examination subsequently carried out in the presence of polymerase, dTTPand dATP gave similar results when compared with the preceding step.This indicated that neither dTTP nor dATP was the next correctnucleotide, because only binary complexes had formed. In contrast, theexamination step carried out in the presence of polymerase, dTTP, dATP,and dCTP indicated a substantial increase in the binding signal comparedwith the preceding step, thereby indicating formation of a ternarycomplex that included dCTP. Formation of the ternary complex aftercontacting the reagent that included dCTP identified dCTP as the nextcorrect base. Finally, the examination step carried out in the presenceof polymerase, dTTP, dATP, dCTP, and dGTP did not indicate any furtherincrease in binding signal compared with the preceding step, asexpected. Thus, inclusion of the non-cognate dGTP did not change thebinding signal once the ternary complex had formed. Notably, theprocedure used to obtain the results presented in FIG. 4 did not includeany nucleotide in the 5 second step ordinarily used for incorporatingthe next correct nucleotide. Accordingly, there was no increase in thebinding signal during that step. The reaction interval is presented inFIG. 4 to illustrate the workflow that includes the incorporationreaction.

Discoveries described above, made using label-free systems, were nextapplied to sequencing systems that rely on exogenous detectable labels.In certain particular instances, systems employing one or morefluorescent polymerases were used to identify cognate nucleotide withoutincorporation. Polymerase used in illustrative examples was uniformlyfluorescent in the presence and absence of added nucleotide. Althoughconformationally sensitive fluorescent polymerases can be used in theprocedure, there is no requirement that this is the case. In certainembodiments, the detectably labeled polymerase does not producesubstantially different signals when bound to primed template nucleicacid in the presence or absence of cognate nucleotide.

Preferably, the polymerases used in separate steps for contacting primedtemplate nucleic acid in the absence and presence of test nucleotides(i.e., cognate nucleotide candidates) have identical amino acidsequences. For example, the two polymerases can differ only by thepresence of an exogenous detectable label on the polymerase used in thepresence of nucleotides. More particularly, the polymerase used in thesecond contacting step that includes a test nucleotide can be achemically modified version of the polymerase employed in the initialcontacting step that did not include any nucleotide being tested duringa complete cycle of testing four different nucleotides.

Generally speaking, there is a benefit to using the biphasic approachfor polymerase binding in polymerase-based sequencing procedures. Wherepreceding discussions of the biphasic approach applied in connectionwith label-free systems focused on reduction of background signals usingthe same polymerase in the first and second contacting steps, sequencingprocedures employing detectably labeled polymerase preferably employnon-identical polymerases. More particularly, the first reaction mixturepreferably includes a polymerase that lacks an exogenous label to beused for indicating the presence of cognate nucleotide withoutincorporation of that nucleotide. The second reaction mixture, which isused in the second contacting step, includes a polymerase having theexogenous label that will indicate cognate nucleotide binding, againwithout incorporation. Using the biphasic binding technique, the primedtemplate nucleic acid is first contacted with a first reaction mixturethat includes a polymerase lacking a fluorescent label of the polymerasewhich is present in the second reaction mixture, and which contacts theprimed template nucleic acid in the second contacting step.

In certain preferred embodiments, the polymerase used in the secondcontacting step differs from the polymerase used in the first contactingstep by including an exogenous chemical label, such as an exogenousfluorescent label. For example, the polymerase used in the initial stepfor contacting the primed template nucleic acid (i.e., using a firstreaction mixture that does not include a cognate nucleotide candidate)can be a polymerase free of exogenous fluorescent moieties. Accordingly,the first polymerase to contact the primed template nucleic acidpreferably does not substantially contribute to a fluorescent signal.The polymerase provided by the second reaction mixture, which is used inthe second contacting step, preferably includes an exogenous fluorescentlabel that is absent from the polymerase of the first reaction mixture.It is to be understood that identification of a cognate nucleotidewithout incorporation in sequencing-by-binding procedures associate adetectable signal with ternary complex formation.

In procedures that rely on serial addition of different nucleotides(i.e., contacting the primed template with a second nucleotide beforeany complex containing an earlier-added nucleotide has been removed),the polymerase of subsequent steps optionally includes the same label,or a different label (e.g., the same or different fluorescent label).What is important is that the polymerase of the initial contacting step(referred to herein as the first contacting step) is missing thefluorescent label of any subsequent step(s) to be used for indicatingcognate nucleotide binding. Stated differently, in sequencing-by-bindingprocedures employing one or more labeled polymerase to identify acognate nucleotide, the primed template nucleic acid should initiallycontact polymerase in the absence of cognate nucleotide candidates foreach complete cycle of nucleotide interrogation. If four steps ofnucleotide binding are to be investigated, then the initial step, whichpreceded the examination step, should involve contacting primed templatenucleic acid with polymerase in the absence of any of the four testnucleotides.

The biphasic approach advantageously improves results obtained inprocedures employing a single labeled polymerase, or a plurality ofdifferentially labeled polymerases. Improved results arise fromreduction of binding signals due to the presence of polymerase prior tocontacting any nucleotide.

When a particular detectable label is used for indicating cognatenucleotide binding generally (i.e., a single label being used toindicate ternary complex formation), the particular detectable labelshould be absent from the polymerase that contacted the primed templatenucleic acid in the absence of any cognate nucleotide candidate. In thisinstance the same detectably labeled polymerase would be present duringaddition of each different nucleotide, where different nucleotides areadded one at a time. To clarify, the primed template nucleic acid shouldbe contacted initially with a polymerase in the absence of nucleotide,where the polymerase does not include the detectable label to be usedfor indicating ternary complex formation. In a first nucleotide bindingstep, the primed template nucleic acid from the initial step iscontacted with a reaction mixture including the labeled polymerase and afirst nucleotide, in the absence of the polymerase from the initialcontacting step (e.g., the “unlabeled” polymerase). In a secondnucleotide binding step, the primed template nucleic acid from theprevious step is contacted with a reaction mixture including the labeledpolymerase and a second nucleotide, again in the absence of thepolymerase from the initial contacting step. Optionally, the firstnucleotide is included with the second nucleotide in the reactionmixture that includes the labeled polymerase, but not the polymerasefrom the initial contacting step. The process is repeated for the thirdand fourth nucleotides, each time using reaction mixtures progressivelychanging out or adding one nucleotide at a time, where the labeledpolymerase is included, but the polymerase from the initial contactingstep is omitted.

Parallel benefits are realized when using a plurality of differentiallylabeled polymerases to identify cognate nucleotides withoutincorporation. As above, an initial step for contacting the primedtemplate nucleic acid should be performed using a reaction mixture thatincludes polymerase in the absence of any added nucleotide to be testedas a potential cognate nucleotide. Again as above, the polymerase of theinitial contacting step should not harbor the same detectable label asany polymerase used subsequently to indicate cognate nucleotide binding.Next, nucleotide interrogation begins by contacting the primed templatenucleic acid from the previous step with a reaction mixture thatincludes a first labeled polymerase and a first nucleotide to be testedas the cognate nucleotide. Next, the primed template nucleic acid fromthe previous step can be contacted with a reaction mixture that includesa second labeled polymerase and a second nucleotide to be tested as thecognate nucleotide. This reaction includes only the second labeledpolymerase, and does not include either the polymerase of the initialcontacting step, or the first labeled polymerase. In this way,nucleotide binding can be coded by association with the label on thepolymerase. Next, the primed template nucleic acid from the previousstep can be contacted with a reaction mixture that includes a thirdlabeled polymerase and a third nucleotide to be tested as the cognatenucleotide. This reaction includes only the third labeled polymerase,and does not include the polymerase of the initial contacting step, thefirst labeled polymerase, or the second labeled polymerase. Again,nucleotide binding can be coded by association with the label on thepolymerase. Finally, the primed template nucleic acid from the previousstep can be contacted with a reaction mixture that includes a fourthlabeled polymerase and a fourth nucleotide to be tested as the cognatenucleotide. This reaction includes only the fourth labeled polymerase,and does not include the polymerase of the initial contacting step, thefirst labeled polymerase, the second labeled polymerase, or the thirdlabeled polymerase. Again, nucleotide binding can be coded byassociation with the label on the polymerase. The first, second, third,and fourth polymerases all can harbor distinguishable labels. By thisapproach, a single scan may be performed as part of an examination step.For example, a single fluorescent scan can be carried out to detectternary complexes containing each of the differentially labeledpolymerases.

Disclosed above are materials, compositions, and components that can beused for, can be used in conjunction with, can be used in preparationfor, or are products of the disclosed methods and compositions. It is tobe understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, and that while specific referenceof each various individual and collective combinations and permutationsof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure, including steps in methods using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific method steps or combination ofmethod steps of the disclosed methods, and that each such combination orsubset of combinations is specifically contemplated and should beconsidered disclosed.

Publications cited herein, and the material for which they are cited,are hereby specifically incorporated by reference in their entireties.All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that the headings used herein are fororganizational purposes only and are not meant to limit the descriptionor claims.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

1. A method of determining whether a test nucleotide is the next correctnucleotide comprising a base complementary to the next base in atemplate strand immediately downstream of a primer in a primed templatenucleic acid, said method comprising the steps of: (a) contacting theprimed template nucleic acid with a first reaction mixture thatcomprises a DNA polymerase, thereby forming a complex comprising theprimed template nucleic acid and the polymerase; (b) contacting thecomplex from step (a) with a second reaction mixture that comprises thetest nucleotide, thereby forming a ternary complex comprising the primedtemplate nucleic acid, the polymerase and the test nucleotide; (c)measuring binding of the primed template nucleic acid to the DNApolymerase at one or more points during each of steps (a) and (b),respectively, but without chemical incorporation of the test nucleotideinto the primer; and (d) determining whether the test nucleotide is thenext correct nucleotide using the measured binding results from step(c).
 2. The method of claim 1, wherein the test nucleotide is a nativenucleotide that does not comprise any added fluorescent label, andwherein step (c) does not comprise measuring differences in fluorescenceor absorbance signals from a conformationally sensitive dye that changesoptical properties as the result of the DNA polymerase binding to thetest nucleotide.
 3. The method of claim 1, wherein the first reactionmixture does not comprise the test nucleotide.
 4. The method of claim 3,further comprising the step of (e) chemically incorporating the testnucleotide into the primer of the primed template nucleic acid byformation of a phosphodiester bond.
 5. The method of claim 4, furthercomprising repeating steps (a)-(e).
 6. (canceled)
 7. (canceled)
 8. Themethod of claim 1, further comprising the step of (e) replacing thesecond reaction mixture with a third reaction mixture that comprises areversible terminator nucleotide comprising a reversible terminatormoiety, and a DNA polymerase.
 9. The method of claim 8, furthercomprising the step of (f) incorporating the reversible terminatornucleotide into the primer by formation of a phosphodiester bond. 10.The method of claim 9, further comprising the step of (g) removing thereversible terminator moiety from the reversible terminator nucleotidethat incorporated into the primer.
 11. The method of claim 10, furthercomprising repeating steps (a)-(g) at least 50 times. 12.-14. (canceled)15. The method of claim 3, wherein step (c) comprises measuring opticalsignals indicating binding of the primed template nucleic acid to theDNA polymerase and then calculating the difference between the measuredoptical signals to determine whether the measured binding resulting fromstep (b) exceeds the measured binding resulting from step (a).
 16. Themethod of claim 3, wherein step (c) comprises measuring optical signalsindicating binding of the primed template nucleic acid to the DNApolymerase and then calculating the ratio of the measured opticalsignals to determine whether the measured binding resulting from step(b) exceeds the measured binding resulting from step (a).
 17. (canceled)18. (canceled)
 19. The method of claim 3, wherein the test nucleotidedoes not comprise an exogenous fluorescent label.
 20. (canceled)
 21. Themethod of claim 3, wherein the test nucleotide is a nucleotide analog,that comprises an exogenous label that is detected in step (c). 22.-25.(canceled)
 26. The method of claim 1, wherein the DNA polymerase is freeof added fluorescent label.
 27. The method of claim 1, wherein the DNApolymerase is free of added fluorescent label that changes propertiesafter the DNA polymerase binds to a nucleotide. 28.-30. (canceled) 31.The method of claim 1, wherein step (d) comprises determining that thetest nucleotide is the next correct nucleotide if the measured bindingresulting from step (b) exceeds the measured binding resulting from step(a). 32.64. (canceled)
 65. The method of claim 1, wherein step (b)comprises sequentially: (i) contacting the complex from step (a) with areaction mixture that comprises a nucleotide, (ii) removing thenucleotide from contact with the complex, and (iii) repeating (i) and(ii) for different reaction mixtures, each of the different reactionmixtures comprising a different type of nucleotide, wherein one of thedifferent types of nucleotides is the test nucleotide, thereby forming aternary complex comprising the primed template nucleic acid, thepolymerase and the test nucleotide.
 66. The method of claim 1, whereinthe first reaction mixture comprises a nucleotide that is different fromthe test nucleotide.
 67. The method of claim 1, wherein the complexformed in step (a) is a binary complex.